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News Article | September 7, 2016
Site: www.nature.com

All mice were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care accredited animal facility at the University of Pennsylvania or Yale University. Mice were housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by an institutional Animal Care and Use Committee. Male and female mice between 4 and 12 weeks of age were used for all experiments. Littermate controls were used whenever possible. C57BL/6 (wild type) and B6.SJL-Ptprca Pepcb/Boy (B6.SJL) mice were purchased from The Jackson Laboratory. We generated Morrbid-deficient mice and the in cis and in trans double heterozygous mice (Morrbid+/−, Bcl2l11+/−) mice using the CRISPR/Cas9 system as previously described26. In brief, to generate Morrbid-deficient mice, single guide RNAs (sgRNAs) were designed against regions flanking the first and last exon of the Morrbid locus (Extended Data Fig. 1g). Cas9-mediated double-stranded DNA breaks resolved by non-homologous end joining (NHEJ) ablated the intervening sequences containing Morrbid in C57BL/6N one-cell embryos. The resulting founder mice were Morrbid−/+, which were then bred to wild-type C57BL/6N and then intercrossed to obtain homozygous Morrbid-/- mice. One Morrbid-deficient line was generated. To control for potential off-target effects, mice were crossed for at least 5 generations to wild-type mice and then intercrossed to obtain homozygosity. Littermate controls were used when possible throughout all experiments. To generate the in cis and in trans double heterozygous mice (Morrbid+/−, Bcl2l11+/−) mice, we first obtained mouse one-cell embryos from a mating between Morrbid−/− female mice and wild-type male mice. As such, the resulting one-cell embryos were heterozygous for Morrbid (Morrbid+/−). We then micro-injected sgRNAs designed against intronic sequences flanking the second exon of Bcl2l11, which contains the translational start site/codon, into Morrbid−/+ one-cell embryos (Extended Data Fig. 9). Cas9-mediated double-stranded DNA breaks resolved by NHEJ ablated the intervening sequences containing the second exon of Bcl2l11 in Morrbid+/− (C57BL/6N) one-cell embryos, generating founder mice that were heterozygous for both Bcl2l11 and Morrbid (Bcl2l11+/−; Morrbid−/+). Founder heterozygous mice were then bred to wild-type C57BL/6N to interrogate for the segregation of the Morrbid-deficient and Bcl2l11-defient alleles (Extended Data Fig. 9). Pups that segregated such alleles were named in trans and pups that did not segregate were labelled in cis. One line of in cis and in trans double heterozygous mice (Bcl2l11+/−; Morrbid−/+) lines were generated. To control for potential off-target effects, mice were crossed for at least 5 generations to wild-type (C57BL/6N) mice (for in cis) and to Morrbid−/− mice (for in trans) to maintain heterozygosity. To determine genetic rescue, samples from mice containing different permutations of Morrbid and Bcl2l11 alleles (Fig. 4g–j) were analysed in a blinded manner by a single investigator not involved in the breeding or coding of these samples. Cells were isolated from the indicated tissues (blood, spleen, bone marrow, peritoneal exudate, adipose tissue). Red blood cells were lysed with ACK. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. If run live, cells were stained with 7-AAD (7-amino-actinomycin D) to exclude non-viable cells. Otherwise, before fixation, Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Active caspase staining using Z-VAD-FMK (CaspGLOW, eBiosciences) was performed according to the manufacturer's specifications. Apoptosis staining by annexin V+ (Annexin V Apoptosis Detection kit) was performed according to the manufacturer’s recommendations. BrdU staining was performed using BrdU Staining Kit (eBioscience) according to the manufacturer’s recommendations. For BCL2L11 staining, cells were fixed for 15 min in 2% formaldehyde solution, and permeabilized with flow cytometry buffer supplemented with 0.1% Triton X-100. All flow cytometry analysis and cell-sorting procedures were done at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analysers and a BD FACSAria II sorter running FACSDiva software (BD Biosciences). FlowJo software (version 10 TreeStar) was used for data analysis and graphic rendering. All fluorochrome-conjugated antibodies used are listed in Supplementary Table 2. 1 × 106 wild-type and Morrbid-deficient neutrophils sorted from mouse bone marrow were assayed for BCL2L11 protein expression by western blotting (Bim C34C5 rabbit monoclonal antibody, Cell Signaling), as previously described. 2 × 106 wild-type and Morrbid-deficient neutrophils sorted from mouse bone marrow were cross-linked in a 1% formaldehyde solution for 5 min at room temperature while rotating. Crosslinking was stopped by adding glycine (0.2 M in 1 × PBS (phosphate buffered saline)) and incubating on ice for 2 min. Samples were spun at 2500g for 5 min at 4 °C and washed 4 times with 1 × PBS. The pellets were flash frozen and stored at −80 °C. Cells were lysed, and nuclei were isolated and sonicated for 8 min using a Covaris S220 (105 Watts, 2% duty cycle, 200 cycles per burst) to obtain approximately 200–500 bp chromatin fragments. Chromatin fragments were pre-cleared with protein G magnetic beads (New England BioLabs) and incubated with pre-bound anti-H3K27me3 (Qiagen), anti-EZH2 (eBiosciences), or mouse IgG1 (Santa Cruz Biotechnology) antibody-protein G magnetic beads overnight at 4 °C. Beads were washed once in low-salt buffer (20 mM Tris, pH 8.1, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice in high-salt buffer (20 mM Tris, pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), once in LiCl buffer (10 mM Tris, pH 8.1, 1 mM EDTA, 0.25 mM LiCl, 1% NP-40, 1% deoxycholic acid) and twice in TE buffer (10 mM Tris-HCl, pH 8. 0, 1 mM EDTA). Washed beads were eluted twice with 100 μl of elution buffer (1% SDS, 0.1 M NaHCO ) and de-crosslinked (0.1 mg ml−1 RNase, 0.3 M NaCl and 0.3 mg ml−1 Proteinase K) overnight at 65 °C. The DNA samples were purified with Qiaquick PCR columns (Qiagen). qPCR was carried out on a ViiA7 Real-Time PCR System (ThermoFisher) using the SYBR Green detection system and indicated primers. Expression values of target loci were directly normalized to the indicated positive control loci, such as MyoD1 for H3K27me3 and EZH2 ChIP analysis, and Actb for Pol II ChIP analysis. ChIP–qPCR primer sequences are listed in Supplementary Table 1. 50,000 wild-type and knockout cells, in triplicate, were spun at 500g for 5 min at 4 °C, washed once with 50 μl of cold 1× PBS and centrifuged in the same conditions. Cells were resuspended in 50 μl of ice-cold lysis buffer (10 mM Tris-HCl, pH7.4, 10 mM NaCl, 3 mM MgCl , 0.1% IGEPAL CA-630). Cells were immediately spun at 500g for 10 min at 4 °C. Lysis buffer was carefully pipetted away from the pellet, which was then resuspended in 50 μl of the transposition reaction mix (25 μl 2× TD buffer, 2.5 μl Tn5 Transposase (Illumina), 22.5 μl nuclease-free water) and then incubated at 37 °C for 30 min. DNA purification was performed using a Qiagen MinElute kit and eluted in 12 μl of Elution buffer (10 mM Tris buffer, pH 8.0). To amplify library fragments, 6 μl of the eluted DNA was mixed with NEBnext High-Fidelity 2× PCR Master Mix, 25 μM of customized Nextera PCR primers 1 and 2 (Supplementary Table 1), 100x SYBR Green I and used in PCR as follow: 72 °C for 5 min; 98 °C for 30 s; and thermocycling 4 times at 98 °C for 10 s; 63 °C for 30 s; 72 °C for 1 min. 5 μl of the 5 cycles PCR amplified DNA was used in a qPCR reaction to estimate the additional number of amplification cycles. Libraries were amplified for a total of 10–11 cycles and were then purified using a Qiagen PCR Cleanup kit and eluted in 30 μl of Elution buffer. The libraries were quantified using qPCR and bioanalyser data, and then normalized and pooled to 2 nM. Each 2 nM pool was then denatured with a 0.1 N NaOH solution in equal parts then further diluted to form a 20 pM denatured pool. This pool was then further diluted down to 1.8 pM for sequencing using the NextSeq500 machine on V2 chemistry and sequenced on a 1 × 75 bp Illumina NextSeq flow cell. ATAC sequencing cells was done on Illumina NextSeq at a sequencing depth of ~40–60 million reads per sample. Libraries were prepared in triplicates. Raw reads were deposited under GSE85073. 2 × 75 bp paired-end reads were mapped to the mouse mm9 genome using ‘bwa’ algorithm with ‘mem’ option. Only reads that uniquely mapped to the genome were used in subsequent analysis. Duplicate reads were eliminated to avoid potential PCR amplification artifacts and to eliminate the high numbers of mtDNA duplicates observed in ATAC–seq libraries. Post-alignment filtering resulted in ~26–40 million uniquely aligned singleton reads per library and the technical replicates were merged into one alignment BAM file to increase the power of open chromatin signal in downstream analysis. Depicted tracks were normalized to total read depth. ATAC–seq enriched regions (peaks) in each sample was identified using MACS2 using the below settings: 10 × 106 wild-type and knockout mice neutrophils were cross-linked in a 1% formaldehyde solution for 10 min at room temperature while rotating. Crosslinking was stopped by adding glycine (0.2 M in 1 × PBS) and incubating on ice for 2 min. Samples were spun at 2500g for 5 min at 4 °C and washed 4 times with 1× PBS. The pellets were flash frozen and stored at −80 °C. Cells were lysed and sonicated (Branson Sonifier 250) for 9 cycles (30% amplitude; time, 20 s on, 1 min off). Lysates were spun at 18,400g for 10 min at 4 °C and resuspended in 3 ml of lysis buffer. A sample of 100 μl was kept aside as input and the rest of the samples were divided by the number of antibodies to test. Chromatin immunoprecipitation was performed with 10 μg of antibody-bound beads (anti-H3K27ac, H3K4me3, H3K4me1, H3K36me3 (Abcam) and anti-rabbit IgG (Santa Cruz), Dynal Protein G magnetic beads (Invitrogen)) and incubated overnight at 4 °C. Bead-bound DNA was washed, reverse cross-linked and eluted overnight at 65 °C, shaking at 950 r.p.m. Beads were removed using a magnetic stand and eluted DNA was treated with RNase A (0.2 μg μl−1) for 1 h at 37 °C shaking at 950 r.p.m., then with proteinase K (0.2 μg μl−1) for 2 h at 55 °C. 30 μg of glycogen (Roche) and 5 M of NaCl were adding to the samples. DNA was extracted with 1 volume of phenol:chlorofrom:isoamyl alcohol and washed out with 100% ethanol. Dried DNA pellets were resuspended in 30 μl of 10 mM Tris HCl, pH 8.0, and DNA concentrations were quantified using Qubit. Starting with 10 ng of DNA, ChIP–seq libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems, Inc.) with 10 cycles of PCR. The libraries were quantified using qPCR and bioanalyser data then normalized and pooled to 2 nM. Each 2 nM pool was then denatured with a 0.1 N NaOH solution in equal parts then further diluted to form a 20 pM denatured pool. This pool was then further diluted down to 1.8 pM for sequencing using the NextSeq500 machine on V2 chemistry and sequenced on a 1 × 75 bp Illumina NextSeq flow cell. ChIP sequencing was done on an Illumina NextSeq at a sequencing depth of ~30–40 million reads per sample. Raw reads were deposited under GSE85073. 75 bp single-end reads were mapped to the mouse mm9 genome using ‘bowtie2’ algorithm. Duplicate reads were eliminated to avoid potential PCR amplification artifacts and only reads that uniquely mapped to the genome were used in subsequent analysis. Depicted tracks were normalized to control IgG input sample. ChIP–seq-enriched regions (peaks) in each sample was identified using MACS2 using the below settings: 107 immortalized BMDMs were collected by trypsinization and resuspended in 2 ml PBS, 2 ml nuclear isolation buffer (1.28 M sucrose; 40 mM Tris-HCl, pH 7.5; 20 mM MgCl ; 4% Triton X-100), and 6 ml water on ice for 20 min (with frequent mixing). Nuclei were pelleted by centrifugation at 2,500g for 15 min. Nuclear pellets were resuspended in 1 ml RNA immunoprecipitation (RIP) buffer (150 mM KCl, 25 mM Tris, pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40; 100 U ml−1 SUPERaseIn, Ambion; complete EDTA-free protease inhibitor, Sigma). Resuspended nuclei were split into two fractions of 500 μl each (for mock and immunoprecipitation) and were mechanically sheared using a dounce homogenizer. Nuclear membrane and debris were pelleted by centrifugation at 15,800g. for 10 min. Antibody to EZH2 (Cell Signaling 4905S; 1:30) or normal rabbit IgG (mock immunoprecipitation, SantaCruz; 10 μg) were added to supernatant and incubated for 2 hours at 4 °C with gentle rotation. 25 μl of protein G beads (New England BioLabs S1430S) were added and incubated for 1 hour at 4 °C with gentle rotation. Beads were pelleted by magnetic field, the supernatant was removed, and beads were resuspended in 500 μl RIP buffer and repeated for a total of three RIP buffer washes, followed by one wash in PBS. Beads were resuspended in 1 ml of Trizol. Co-precipitated RNAs were isolated, reverse-transcribed to cDNA, and assayed by qPCR for the Hprt and Morrbid-isoform1. Primer sequences are listed in Supplementary Table 1. EZH2 PAR–CLIP dataset (GSE49435) was analysed as previously described22. Adapter sequences were removed from total reads and those longer than 17 bp were kept. The Fastx toolkit was used to remove duplicate sequences, and the resulting reads were mapped using BOWTIE allowing for two mismatches. The four independent replicates were pooled and analysed using PARalyzer, requiring at least two T→C conversions per RNA–protein contact site. lncRNAs were annotated according to Ensemble release 67. 13 × 106 wild-type bone marrow derived mouse eosinophils were fixed with 1% formaldehyde for 10 minutes at room temperature, and quenched with 0.2 M glycine on ice. Eosinophils were lysed for 3–4 hours at 4 °C (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 1% Triton X-100, 1× Roche complete protease inhibitor) and dounce-homogenized. Lysis was monitored by Methyl-green pyronin staining (Sigma). Nuclei were pelleted and resuspended in 500 μl 1.4× NEB3.1 buffer, treated with 0.3% SDS for one hour at 37 °C, and 2% Triton X-100 for another hour at 37 °C. Nuclei were digested with 800 units BglII (NEB) for 22 hours at 37 °C, and treated with 1.6% SDS for 25 minutes at 65 °C to inactivate the enzyme. Digested nuclei were suspended in 6.125 ml of 1.25× ligation buffer (NEB), and were treated with 1% Triton X-100 for one hour at 37 °C. Ligation was performed with 1,000 units T4 DNA ligase (NEB) for 18 hours at 16 °C, and crosslinks were reversed by proteinase K digestion (300 μg) overnight at 65 °C. The 3C template was treated with RNase A (300 μg), and purified by phenol-chloroform extraction. Digested and undigested DNA were run on a 0.8% agarose gel to confirm digestion. To control for PCR efficiency, two bacterial artificial chromosomes (BACs) spanning the region of interest were combined in equimolar quantities and digested with 500 units BglII at 37 °C overnight. Digested BACs were ligated with 100 units T4 Ligase HC (Promega) in 60 μl overnight at 16 °C. Both BAC and 3C ligation products were amplified by qPCR (Applied Biosystems ViiA7) using SYBR fast master mix (KAPA biosystems). Products were run side by side on a 2% gel, and images were quantified using ImageJ. Intensity of 3C ligation products was normalized to intensity of respective BAC PCR product. Mice were infected with 30,000 CFUs of Listeria monocytogenes (strain 10403s, obtained as a gift from E. J. Wherry) intravenously (i.v.). Mice were weighed and inspected daily. Mice were analysed at day 4 of infection to determine the CFUs of L. monocytogenes present in the spleen and liver. Papain was purchased from Sigma Aldrich and resuspended in at 1 mg ml−1 in PBS. Mice were intranasally challenged with 5 doses of 20 μg papain in 20 μl of PBS or PBS alone every 24 hours. Mice were killed 12 hours after the last challenge. Bronchoalveolar lavage was collected in two 1 ml lavages of PBS. Cellular lung infiltrates were collected after 1 hour digestion in RPMI supplemented with 5% FCS, 1 mg ml−1 collagenase D (Roche) and 10 μg ml−1 DNase I (Invitrogen) at 37 °C. Homogenates were passed through a cell strainer and infiltrates separated with a 27.5%, Optiprep gradient (Axis-Shield) by centrifugation at 1,175g for 20 min. Cells were removed from the interface and treated with ACK lysis buffer. Congenic C57BL/6 (wild-type) bone marrow expressing CD45.1 and CD45.2 and Morrbid-deficient bone marrow expression CD45.2 was mixed in a 1:1 ratio and injected into C57BL/6 hosts irradiated twice with 5 Gy 3 hours apart that express CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ). Mice were analysed between 4–9 weeks after injection. Bone marrow was isolated and cultured as previously described9. Briefly, unfractionated bone marrow cells were cultured with 100 ng ml−1 stem cell factor (SCF) and 100 ng ml−1 FLT3-ligand (FLT3-L). At day 4, the media was replaced with media containing 10 ng ml−1 interleukin (IL-5). Mature bone-marrow-derived eosinophils were analysed between day 10–14. Bone marrow cells were isolated and cultured in media containing recombinant mouse M-CSF (10 ng ml−1) for 7–8 days. On day 7–8, cells were re-plated for use in experimental assays. Bone-marrow-derived macrophages were stimulated with LPS (250 ng ml−1) for the indicated periods of time. Briefly, 40 × 107 Immortalized bone-marrow-derived macrophages were fixed with 40 ml of 1% glutaraldehyde for 10 min at room temperature. Crosslinking was quenched with 0.125 M glycine for 5 min. Cells were rinsed with PBS, pelleted for 4 min at 2,000g, snap-frozen in liquid nitrogen, and stored at −80 °C. Cell pellets were thawed at room temperature and resuspended in 800 μl of lysis buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, complete protease inhibitor (Roche), 0.1 U ml−1 Superase In (Life Technologies)). Cell suspension was sonicated using a Covaris S220 machine (Covaris; 100 W, duty factor 20%, 200 cycles per burst) for 60 minutes until DNA was in the size range of 100–500 bp. After centrifugation for 5 min at 16100 g at 4 °C, the supernatant was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C. 1 ml of chromatin was diluted in 2 ml hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris HCl, pH 7.0, 1 mM EDTA, 15% formamide) and input RNA and DNA aliquots were removed. 100 pmoles of probes (Supplementary Table 1) were added and mixed by rotation at 37 °C for 4 h. Streptavidin paramagnetic C1 beads (Invitrogen) were equilibrated with lysis buffer. 100 μl washed C1 beads were added, and the entire reaction was mixed for 30 min at 37 °C. Samples were washed five times with 1 ml of washing buffer (SSC 2×, 0.5% SDS and fresh PMSF). 10% of each sample was removed from the last wash for RNA isolation. RNA aliquots were added to 85 μl RNA PK buffer, pH 7.0, (100 mM NaCl, 10 mM TrisCl, pH 7.0, 1 mM EDTA, 0.5% SDS, 0.2 U μl−1 proteinase K) and incubated for 45 min with end-to-end shaking. Samples were spun down, and boiled for 10 min at 95 °C. Samples were chilled on ice, added to 500 μl TRizol, and RNA was extracted according to the manufacturer’s recommendations. Equal volume of RNA was reverse-transcribed and assayed by qPCR using Hprt and Morrbid-exon1-1 primer sets (Supplementary Table 1). DNA was eluted from remaining bead fraction twice using 150 μl DNA elution buffer (50 mM NaHCO , 1%SDS, 200 mM NaCl, 100 μg ml−1 RNase A, 100 U ml−1 RNase H) incubated for 30 min at 37 °C. DNA elutions were combined and treated with 15 μl (20 mg ml−1) Proteinase K for 45 min at 50 °C. DNA was purified using phenol:chloroform:isoamyl and assayed by qPCR using the indicated primer sequences (Supplementary Table 1). shRNAs of indicated sequences (Supplementary Table 1) were cloned into pGreen shRNA cloning and expression lentivector. Psuedotyped lentivirus was generated as previously described, and 293T cells were transfected with a packaging plasmid, envelop plasmid, and the generated shRNA vector plasmid using Lipofectamine 2000. Virus was collected 14–16 h and 48 h after transfection, combined, 0.4-μm filtered, and stored at −80 °C. For generation of in vivo BM chimaeras, virus was concentrated 6 times by ultracentrifugation using an Optiprep gradient (Axis-Shield). For transduced BM-derived eosinophils, cultured BM cells on day 3 of previously described culture conditions were mixed 1:1 with indicated lentivirus and spinfected for 2 h at 260g at 25 °C with 5 μg ml−1 polybrene. Cultures were incubated overnight at 37 °C, and media was exchanged for IL-5 containing media at day 4 of culture as previously described9. Cells were sorted for GFP+ cells on day 5 of culture, and then cultured as previously described for eosinophil generation. Cells were assayed on day 11 of culture. For transduced in vivo BM chimaeras, BM cells were cultured at 2.5 × 106 cells per ml in mIL-3 (10 ng ml−1), mIL-6 (5 ng ml−1) and mSCF (100 ng ml−1) overnight at 37 °C. Culture was readjusted to 2 ml at 2.5 × 106 cells per ml in a 6-well plate, and spinfected for 2 h at 260g at 25 °C with 5 μg ml−1 polybrene. Cells were incubated overnight at 37 °C. On the day before transfer, recipient hosts were irradiated twice with 5 Gy 3 hours apart. Mice were analysed between 4 and 5 weeks following transfer. Bone marrow-derived macrophages (BMDMs) were transfected with pooled Morrbid or scrambled locked nucleic acid (LNA) antisense oligonucleotides of equivalent total concentrations using Lipofectamine 2000. Morrbid LNA pools contained Morrbid LNA 1-4 sequences at a total of 50 or 100 nM (Supplementary Table 1). After 24 h, the transfection media was replaced. The BMDMs were incubated for an additional 24 h and subsequently stimulated with LPS (250 ng ml−1) for 8−12 h. Eosinophils were derived from mouse BM as previously described. On day 12 of culture, 1 × 106 to 2 × 106 eosinophils were transfected with 50 nm of Morrbid LNA 3 or scrambled LNA (Supplementary Table 1) using TransIT-oligo according to manufacturer’s protocol. RNA was extracted 48 h after transfection. Guide RNAs (gRNAs) targeting the 5’ and 3’ flanking regions of the Morrbid promoter were cloned into Cas9 vectors pSPCas9(BB)-2A-GFP(PX458) (Addgene plasmid 48138) and pSPCas9(BB)-2A-mCherry (a gift from the Stitzel lab, JAX-GM) respectively. gRNA sequences are listed in Supplementary Table 1. The cloned Cas9 plasmids were then transfected into RAW 264.7, a mouse macrophage cell line using Lipofectamine 2000, according to manufacturer’s protocol. Forty–eight hours post transfection the double positive cells expressing GFP and mcherry, and the double negative cells lacking GFP and mcherry were sorted. The bulk sorted cells were grown in a complete media containing 20% FBS, assayed for deletion by PCR, as well as for Morrbid and Bcl2l11 transcript expression by qPCR. BM-derived eosinophils, or neutrophils or Ly6Chi monocytes sorted from mouse BM, were rested for 4–6 hours at 37 °C in complete media. Cells were subsequently stimulated with IL-3 (10 ng ml−1, Biolegend), IL-5 (10 ng ml−1, Biolegend), GM-CSF (10 ng ml−1, Biolegend), or G-CSF (10 ng ml−1, Biolegend) for 4–6 h. RNA was collected at each time-point using TRIzol (Life Technologies). Wild-type and Bcl2l11−/− BM-derived eosinophils were generated as previously described9. On day 8 of culture, the previously described IL-5 media was supplemented with the indicated concentrations of the EZH2-specific inhibitor GSK126 (Toronto Research Chemicals). Media was exchanged for fresh IL-5 GSK126 containing media every other day. Cells were assayed for numbers and cell death by flow cytometry every day for 6 days following GSK126 treatment. Total RNA was extracted from TRIzol (Life Technologies) according to the manufacturer’s instructions. Gycogen (ThermoFisher Scientific) was used as a carrier. Isolated RNA was quantified by spectophotemetry, and RNA concentrations were normalized. cDNA was synthesized using SuperScript II Reverse Transcriptase (ThermoFisher Scientific) according to the manufacturer’s instructions. Resulting cDNA was analysed by SYBR Green (KAPA SYBR Fast, KAPABiosystems) or Taqman-based (KAPA Probe Fast, KAPABiosystems) using indicated primers. Primer sequences are listed in Supplementary Table 1. All reactions were performed in duplicate using a CFX96 Touch instrument (BioRad) or ViiA7 Real-Time PCR instrument (ThermoFischer Scientific). Reads generated from mouse (Gr1+) granulocytes (previously published GSE53928), human neutrophils (previously published GSE70068), and bovine peripheral blood leukocytes (previously published GSE60265) were filtered, normalized, and aligned to the corresponding host genome. Reads mapping around the Morrbid locus were visualized. For visualization of the high level of Morrbid expression in short-lived myeloid cells, reads from sorted mouse eosinophils (previously published GSE69707), were filtered, aligned to mm9, normalized using RPKM, and gene expression was plotted in descending order. For each human sample corresponding to the indicated stimulation conditions, the number of reads mapping to the human MORRBID locus per total mapped reads was determined. For conservation across species, the genomic loci and surrounding genomic regions for the species analysed were aligned with mVista and visualized using the rankVista display generated with mouse as the reference sequence. Green highlights annotated mouse exonic regions and corresponding regions in other indicated species. Single molecule RNA fluorescence in situ hybridization (FISH) was performed as previously described. A pool of 44 oligonucleotides (Biosearch Technologies) were labelled with Atto647N (Atto-Tec). For validation purposes, we also labelled subsets consisting of odd and even numbered oligonucleotides with Atto647N and Atto700, respectively, and looked for colocalization of signal. We designed the oligonucleotides using the online Stellaris probe design software. Probe oligonucleotide sequences are listed in Supplementary Table 1. Thirty Z-sections with a 0.3-μm spacing were taken for each field of view. We acquired all images using a Nikon Ti-E widefield microscope with a 100× 1.4NA objective and a Pixis 1024BR cooled CCD camera. We counted the mRNA in each cell by using custom image processing scripts written in MATLAB. For nuclear and cytoplasmic fractionation, 5 × 106 BMDMs were stimulated with 250 ng ml−1 LPS for 4 hours. Cells were collected and washed once with cold PBS. Cells were pelleted, resuspended in 100 μl cold NAR A buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1× complete EDTA-free protease inhibitor, Sigma; 1 mM DTT, 20 mM β-glycerophasphate, 0.1 U μl−1 SUPERaseIn, Life Technologies), and incubated at 4 °C for 20 min. 10 μl 1% NP-40 was added, and cells were incubated for 3 min at room temperature. Cells were vortexed for 30 seconds, and centrifuged at 3,400g. for 1.5 min at 4 °C. Supernatant was removed, centrifuged at full speed for 90 min at 4 °C, and remaining supernatant was added to 500 μl Trizol as the cytoplasmic fraction. The original pellet was washed 4 times in 100 μl NAR A with short spins of 6,800g. for 1 min. The pellet was resuspended in 50 μl NAR C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1× complete EDTA-free protease inhibitor, Sigma, 1 mM DTT, 20mM β-glycerophasphate, 0.1 U μl−1 SUPERaseIn, Life Technologies). Cells were vortexed every 3 min for 10 s for a total of 20 min at 4 °C. The sample was centrifuged at maximum speed for 20 min at room temperature. Remaining supernatant was added to 500 μl Trizol as the nuclear fraction. Equivalent volumes of cytoplasmic and nuclear RNA were converted to cDNA using gene specific primers and Super Script II RT (Life Technologies). Fraction was assessed by qPCR for Morrbid-exon1-1 and other known cytoplasmic and nuclear transcripts. Primer sequences are listed in Supplementary Table 1. For cytoplasmic, nuclear, and chromatin fractionation, cell fractions 5 × 106 to 10 × 106 immortalized macrophages were activated with 250 ng ml−1 LPS (Sigma) for 6 hours at 37 °C. Cells were washed 2× with PBS, and then resuspended in 380 μl ice-cold HLB (50 mM Tris-HCl, pH7.4, 50 mM NaCl, 3 mM MgCl , 0.5% NP-40, 10% glycerol), supplemented with 100 U SUPERase In RNase Inhibitor (Life Technologies). Cells were vortexed 30 s and incubated on ice for 30 min, followed by a final 30 s vortex and centrifugation at 4 °C for 5 min × 1000g. Supernatant was collected as the cytoplasmic fraction. Nuclear pellets were resuspended by vortexing in 380 μl ice-cold MWS (50 mM Tris-HCl, pH7.4, 4 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40) supplemented with 100 U SUPERase in RNase Inhibitor. Nuclei were lysed on ice for 10 min, vortexed for 30 s, and incubated on ice for 10 more min to complete lysis. Chromatin was pelleted by centrifugation at 4 °C for 5 min × 1000g. Supernatant was collected as the nucleoplasmic fraction. RNA was collected as described previously and cleaned up using the RNeasy kit (Qiagen). Equivalent volumes of cytoplasmic, nucleoplasmic, and chromatin-associated RNA were converted to cDNA using random hexamers and Super Script III RT (Life Technologies). Fraction was assessed by qPCR for Morrbid-exon1-2 and other known cytoplasmic and nuclear transcripts. Primer sequences are listed in Supplementary Table 1. Morrbid cDNA was cloned into reference plasmid (pCDNA3.1) containing a T7 promoter. The plasmid was linearized and Morrbid RNA was in vitro transcribed using the MEGAshortscript T7 kit (Life Technologies), according to the manufacturer’s recommendations, and purified using the MEGAclear kit (Life Technologies). RNA was quantified using spectrophotometry and serial dilutions of Morrbid RNA of calculated copy number were spiked into Morrbid-deficient RNA isolated from Morrbid-deficient mouse spleen. Samples were reverse transcribed in parallel with wild-type-sorted neutrophil RNA and B-cell RNA isolated from known cell number using gene-specific Morrbid primers, and the Morrbid standard curve and wild-type neutrophils and B cells were assayed using qPCR with Morrbid-exon 1 primer sets (Supplementary Table 1) Cohorts of mice were given a total of 4 mg bromodeoxyuridine (BrdU; Sigma Aldrich) in 2 separate intraperitoneal (i.p.) injections 3 h apart and monitored over the subsequent 5 days, unless otherwise noted. For analysis cells were stained according to manufacturer protocol (BrdU Staining Kit, ebioscience; anti-BrdU, Biolgend). A one-phase exponential curve was fitted from the peak labelling frequency to 36 h after peak labelling within each genetic background, and the half-life was determined from this curve. Study subjects were recruited and consented in accordance with the University of Pennsylvania Institutional Review Board. Peripheral blood was separated by Ficoll–Paque density gradient centrifugation, and the mononuclear cell layer and erythrocyte/granulocyte pellet were isolated and stained for fluorescence-associated cell sorting as previously described. Neutrophils (live, CD16+F4/80intCD3−CD14−CD19−), eosinophils (live, CD16−F4/80hiCD3−CD14−CD19−), T cells (live, CD3+CD16−), monocytes (live, CD14+CD3−CD16−CD56−), natural killer (NK) cells (live, CD56+CD3−CD16−CD14−), B cells (live, CD19+CD3−CD16−CD14−CD56−). Samples from human subjects were collected on NIAID IRB-approved research protocols to study eosinophilic disorders (NCT00001406) or to provide controls for in vitro research (NCT00090662). All participants gave written informed consent. Eosinophils were purified from peripheral blood by negative selection and frozen at –80 oC in TRIzol (Life Technologies). Purity was >97% as assessed by cytospin. RNA was purified according to the manufacturer’s instructions. Expression analysis by qPCR was performed in a blinded manner by an individual not involved in sample collection or coding of these of these samples. Plasma IL-5 levels were measured by suspension array in multiplex (Millipore). The minimum detectable concentration was 0.1 pg ml−1. RAW 264.7 cells were obtained from ATCC and were not authenticated, but were tested for mycoplasma contamination biannually. Immortalized C57/B6 macrophages were obtained as a generous gift from I. Brodsky. These cells were not authenticated, but were tested for mycoplasma contamination biannually. Samples sizes were estimated based on our preliminary phenotyping of Morrbid-deficient mice. Preliminary cell number analysis of eosinophils, neutrophils, and Ly6Chi monocytes suggested that there were very large differences between wild-type and Morrbid-deficient samples, which would allow statistical interpretation with relatively small numbers and no statistical methods were used to predetermine sample size. No animals were excluded from analysis. All experimental and control mice and human samples were run in parallel to control for experimental variability. The experiments were not randomized. Experiments corresponding to Fig. 3g–i and Fig. 4g–j were performed and analysed in a single-blinded manner. All other experiments were not blinded to allocation during experiments and outcome assessment. Correlation was determined by calculating the Spearman correlation coefficient. Half-life was estimated by calculating the one-phase exponential decay constant from the peak of labelling frequency to 36 h after peak labelling. P values were calculated using a two-way t-test, Mann–Whitney U-test, one-way ANOVA with Tukey post-hoc analysis, Kaplan–Meier Mantel–Cox test, and false discovery rate (FDR) as indicated. FDR was calculated using trimmed mean of M-values (TMM)-normalized read counts and the DiffBind R package as described in Extended Data Fig. 7c, d. All error bars indicate mean plus and minus the standard error of mean (s.e.m.).


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Lapatinib, PLX-4032, trametenib, tarceva, ABT-199 and ABT-263 were purchased from Selleck-chem; QVD-OPh from Sigma; MG132 from Calbiochem; idarubicin and araC from Pharmacia and Upjohn. A-1210477 was made according to published methods26. Synthesis and characterization of S63845 is provided in the Supplementary Methods. Owing to light sensitivity, S63845 was stored in the dark. Following the previously published structure of MCL1 (PDB ID4WGI)43, a construct was designed with residues 173–321 of human MCL1 as a C-terminal fusion with maltose binding protein (MBP). In addition to the surface entropy-reducing (SER) mutations in MCL1 (K194A, K197A and R201A (ref. 43)), we also introduced E198A, E199A and K265A mutations into MBP (ref. 44). The plasmid encoding the MBP–MCL1 fusion protein was transformed into BL21(DE3)pLysS bacteria. A single colony was used to inoculate 5 ml terrific broth (Fisher BioReagents, (BP2468-2)) containing kanamycin and chloramphenicol at 100 μg ml−1 and 34 μg ml−1, respectively. After 3 h growth at 37 °C, the 5 ml culture was used to inoculate 2 l terrific broth containing the same antibiotics. At an OD of 0.7, the temperature was reduced to 18 °C before induction of MBP–MCL1 protein expression by addition of IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation. Harvested cells were resuspended in 3 volumes of 20 mM Tris–HCl pH 7.4, 200 mM NaCl, 2 mM EDTA, 1 mM DTT and lysed by passing three times through an emulsiflex-C5 (Avestin). The lysate was clarified by centrifugation at 40,000 g, at 4 °C, for 60 min and applied to a 5-ml MBPTrap column (GE Healthcare). The MBP–MCL1 fusion protein was eluted in 20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2 mM EDTA, 1 mM DTT, 10 mM maltose and further purified by size exclusion chromatography in 20 mM HEPES, 100 mM NaCl and 1 mM DTT. Protein eluted as a monomer was concentrated and used in crystallization studies. Apo crystals were grown at a concentration of 34 mg ml−1 (20 mM HEPES pH 7.5, 150 mM NaCl and 2 mM DTT) by the sitting drop vapour diffusion. 2 μl of the protein solution was mixed with 2 μl of the crystallization reservoir (25% PEG 3350, 0.2 M magnesium formate, 1 mM maltose) in a sitting drop plate. The plate was incubated at 284 K and suitable rod-like crystals appeared overnight. Individual crystals were harvested from the crystallization drops and transferred to a drop containing 4.5 μl of the crystallization reservoir solution plus 0.5 μl of S63845 (20 mM in DMSO). The mixture was incubated for 72 h at 284 K. Crystals were flash frozen in liquid nitrogen after cryoprotection using crystallization reservoir plus 20% ethylene glycol. Diffraction data were collected at the Soleil Synchrotron (France) on a beamline Proxima1 and were processed and scaled using XDS (ref. 45). The structure was solved by molecular replacement using MOLREP (ref. 46), using another crystal structure of an MBP–MCL1 fusion protein43. The data were subsequently refined using REFMAC5 (ref. 47). Interactive graphical model building was carried out with COOT. The ligand was clearly defined by the initial electron density maps. The progress of the refinement was assessed using R and the conventional R factor. Once refinement was completed, the structures were validated using various programs from the CCP4i package, CCP4. Statistic parameters are detailed in Extended Table 1. Fluorescence polarization assays were carried out in black-walled, flat-bottomed, low-binding, 384-well plates (Corning) in buffer A (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20 pH 7.4 and 5% DMSO) in the presence of 10 nM fluorescein-PUMA (3-(((3′,6′-dihydroxy-3-oxo-3H-spiro(2-benzofuran-1,9′-xanthene)-5-yl)carbamothioyl)amino)-N-(6-oxohexyl)propanamide-AREIGAQLRRMADDLNAQY, from the polypeptide group, France). Final concentrations of MCL1, BCL-2 and BCL-X proteins were 10, 10 and 20 nM, respectively. The assay plates were incubated for 2 h at room temperature and the fluorescence polarization was measured on a Synergy 2 reader (exitation, 528 nm; emission, 640 nm; cut-off, 510 nm). The binding of increasing doses of the compound was expressed as a percentage reduction in mP compared to the window established between the ‘DMSO only’ and ‘total inhibition’ control (30 μM PUMA). The inhibitory concentrations of the drugs that gave a 50% reduction in mP (IC ) were determined, from 11-point dose response curves, in XL-Fit using a 4-parameter logistic model (Sigmoidal dose–response model). The K was subsequently calculated as described in ref. 48. All SPR measurements were performed on a BIAcore T200 instrument (BIAcore GE Healthcare). Direct binding experiments were performed at 20 °C on Series S NTA chips. 10 mM HEPES pH 7.4, 175 mM NaCl, 25 μM EDTA, 1 mM TCEP, 0.01% P20 and 1% DMSO was used as a running buffer (buffer B). The ligand surface was generated using double His-tagged proteins essentially as described in refs 49, 50. Serial dilutions of the compound in buffer B were injected over the protein surface. All sample measurements were performed at a flow rate of 30 μlper min (injection time 120 s, dissociation time 360 s). The sensor surface was regenerated by consecutive injections of 0.35 M EDTA pH 8.0 with 0.1 mg/ml−1 trypsin, 0.5 M imidazole and 45% DMSO (60 s, 15 μl per min). Data processing was performed using BIAevaluation 2.1 (BIAcore GE Healthcare Bio-Sciences Corp) software. Sensorgrams were double referenced before global fitting of the concentration series to a 1:1 binding model. Affinity determination by competition in solution experiments were performed at 30 °C in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 1 mM TCEP, 2% glycerol, 0.05% P20 and 1% DMSO (buffer C). An MCL1-specific compound was immobilized on Series S CM5 chips by amine coupling as advised in the BIAcore GE Healthcare protocol. Serial dilutions of compounds in buffer B supplemented with fixed concentrations of protein were injected over the generated surface at a flow rate of 15 μl per min for 90 s. Calibration curves were generated using the same procedure by injecting different concentrations of protein over the same sensor chip. Affinity evaluations were performed using the affinity in solution model of BIA evaluation 2.1 (BIAcore GE Healthcare Bio-Sciences Corp) software. Mice were kept in either the Servier Research Institute or the Walter and Eliza Hall Institute (WEHI) specified pathogen-free animal areas for mouse experimental purpose (for Servier, facility license number B78-100-2). The care and use of animals was in accordance with European and national regulations for the protection of vertebrate animals used for experimental and other scientific purposes (directives 86/609 and 2003/65) and the requirements of the Servier Research Institute and WEHI Animal Ethics Committees. Sample sizes were chosen to reach statistical significance, and tumour measurements and all data analysis were performed in a blinded fashion. The Eμ-Myc transgenic mice are kept on a C57BL/6–Ly5.2+ background and have been described previously51. 8–10 week old female SCID mice (for transplantation with human AMO1 and H929 tumour cells) or Swiss Nude mice (Crl:NU(Ico)-Foxn1nu) (for transplantation with human MV4-11 tumour cells) were inoculated with 0.1 ml containing 5 × 106 of the indicated tumour cells subcutaneously into the right flank. The H929 and MV4-11 cells were resuspended in 100% matrigel (BD Biosciences) and the AMO1 cells in a 50:50 mixture of growth medium and matrigel. The width and length of the tumours were measured 2–3 times a week using an electronic caliper. Tumour volume was calculated using the formula: (length × width2)/2. When the tumour volume reached approximately 200 mm3, mice were randomized into different groups; that is, treatment with drug (different concentrations) or vehicle (n = 8 for each group). S63845 was formulated extemporaneously in 25 mM HCl, 20% 2-hydroxy propyl β-cyclo dextrin 20% (Fisher Scientifics) and administrated at the doses and schedules described in the figure legends. Tumour growth inhibition (TGI ) was calculated at the greatest response using the following equation: where day x is the day maximum where the number of animals per group in the control group is sufficient to calculate the TGI (%). For the statistical analysis of differences in tumour volume between treatment groups, a two-way ANOVA with repeated measures on day factor was performed on log-transformed data followed by Dunnett adjustment in order to compare each dose of drug to the control group. A complete tumour regression response was considered for the population with tumours 25 mm3 for at least three consecutive measurements. For ethical reasons, mice carrying tumours exceeding 2,000 mm3 were euthanized. Data are represented as mean of tumour volume ± s.e.m. over time (days) until at least one mouse per cohort had to be killed. Single-cell suspensions of 106 Eμ-Myc lymphoma cell lines (AH15A, AF47A, BRE966, 2253, MRE 721, 560), resuspended in phosphate-buffered saline (PBS), were injected into the tail vein of 8–9 week old female C57BL/6–Ly5.1+ mice. Mice were treated with either vehicle (25 mM HCl, 20% 2-hydroxy propyl β-cyclo dextrin) or 25 mg kg−1 S63845 (reconstituted in vehicle) on days 5–9 after transplant, administered by tail vein injection or, in some incidences when the tails became damaged, by retro-orbital injection. To generate the survival curves of the mice bearing the Eμ-Myc lymphoma cells, mice were killed when deemed unwell by experienced animal technicians. For the toxicity experiments, female C57BL/6–Ly5.1+ mice bearing Eμ-Myc lymphomas or non-tumour bearing C57BL/6–Ly5.1+ mice were killed 4 days after the 5-day drug treatment regimen had been completed (this equated to 13 days after transplantation of the tumour cells in the mice bearing the Eμ-Myc lymphoma cells). For the three mice injected with the AH15A Eμ-Myc lymphoma cells, those treated with vehicle were analysed after only 4 days of treatment because they were deemed too unhealty from the lymphoma to complete their prescribed regimen. For the maximal tolerated dose experiments, 7–8 week old C57BL/6 mice (3 male and 3 female mice in each arm) were treated with a dose of vehicle or S63845 (25 mg per kg, 40 mg per kg, 50 mg per kg or 60 mg per kg body weight) for 5 consecutive days by i.v. tail vein injection or by retro-orbital injection if the tails became damaged. The mice were analysed as they were killed, or for the mice surviving the entire course of treatment, 3 days after the 5-day treatment had been completed. For the initial toxicity studies, sections of spleen, lymph nodes, thymus, ovaries, uterus, kidneys, liver, pancreas, intestines, colon, heart, lung, sternum, backbone and muscle were fixed in 10% formalin and stained with haematoxylin and eosin. The weights of the spleen, thymus, (axillary, brachial and inguinal) lymph nodes, liver and kidneys were recorded. Cells were flushed from the bone marrow (two femurs and one tibia) and single cell suspensions of the spleen, thymus and lymph nodes were generated. The red blood cells were lysed by treatment with 0.168 M ammonium chloride and the white blood cell count was determined using a CASY cell counter (Scharfe System GmbH). All bone marrow or peripheral blood samples from patients with AML were collected after informed consent in accordance with guidelines approved by the Alfred and Royal Melbourne Hospital human research ethics committees. Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare) density-gradient centrifugation, followed by red cell depletion in ammonium chloride (NH Cl) lysis buffer at 37 °C for 10 min. Cells were then resuspended in PBS containing 2% fetal bovine serum (FBS, Sigma). Mononuclear cells were suspended in RPMI-1640 (Gibco) medium containing penicillin and streptomycin (Gibco) and 15% heat-inactivated FBS (Sigma). Normal CD34+ progenitor cells from healthy donors were collected from granulocyte colony stimulating factor (G-CSF)-mobilized blood harvests and purified after Ficoll separation by CD34 positive selection using Miltenyi Biotec micobeads (Miltenyi Biotec. Cat. No. 130-046-703). The research with primary human cells was approved by the Human Research Ethics Committee (HREC) of Alfred Health. AML cells from patients and cells from normal donors were obtained following informed consent processes approved by the HRECs of Alfred Health and Melbourne Health. Colony-forming assays were performed on freshly purified and frozen mononuclear fractions from AML patients or normal cells from G-CSF mobilized normal, healthy donors. Primary cells were cultured in duplicate in 35-mm dishes (Griener-bio) at 104 to 105 cells. Cells were plated in 0.6% agar (Difco): AIMDM 2× (IMDM powder, Invitrogen), supplemented with NaHCO , dextran, penicillin and streptomycin, β-mercaptoethanol and asparagine, FBS (Sigma) at a 2:1:1 ratio of agar:AIMDM:FBS. For optimal growth conditions, all plates contained granulocyte/macrophage colony stimulating factor (100 ng per plate, genzyme), IL-3 (100 ng per plate, R&D Systems), stem cell factor (100 ng per plate, R&D Systems) and erythropoietin (4U per plate, Roche). Cells were cultured for 2–3 weeks in the presence or absence of drugs at 37 °C at 5% CO in a high humidity incubator. After incubation, plates were fixed with 2.5% glutaraldehyde in saline and scored using GelCount (Oxford Optronix). NCI-H929, RS4;11, MV4-11, HCT-116, BT-474, SK-Mel-2, PC-9 and H146 cells were cultured in RPMI 1640 medium, A2058 cells were cultured in DMEM medium and SK-MEL-28 cells were cultured in EMEM medium. All media were supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 10 mM HEPES pH = 7.4, at 37 °C, in 5% CO . For RS4;11 cells the medium was additionally supplemented with 4.5 g l−1 glucose. AMO1 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated FBS, 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 10mM HEPES pH = 7.4. HeLa cells were cultured in DMEM medium (containing 10% heat-inactivated FBS, 10 mM HEPES, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin). Cells were grown at 37 °C in a humidified atmosphere with 5% CO . All of the cell lines were purchased from the ATCC or DSMZ. Human Burkitt lymphoma (BL)-derived cell lines (Rael-BL, Kem-BL, BL2, BL30, BL31, BL41, and Ramos-BL, a gift from A.B. Rickinson and M. Rowe, University of Birmingham, UK) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1 mM glutamine, 1 mM sodium pyruvate, 50 μM α-thiogycerol (Sigma), and 20 nM bathocuproine disulfonic acid (Sigma) in a humidified incubator at 37 °C, 5% CO . The mouse Eμ-Myc lymphoma cell lines (AH15A, AF47A, 2253, BRE966, MRE 721 and 560) were cultured in high-glucose DMEM supplemented with 10% heat-inactivated FBS, 50 μM β-mercaptoethanol (Sigma), 100 μM asparagine (Sigma), 100 U ml−1 penicillin and 100 mg ml−1 streptomycin in a humidified incubator at 37 °C, 10% CO . The myeloma-derived cell lines were purchased from the ATCC, DSMZ or JCRB or provided by the laboratory of A. Spencer (XG1, KMS-26, ANBL6, WL-2 and OCI-MY1) and cultured as recommended by the suppliers at 37 °C in the presence of 5% CO . Bax−/−,Bak−/− H929, KMS-12-PE and AMO1 cells were generated using CRISPR/Cas9 genome editing as described below. HEK293T cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37 °C in the presence of 10% CO . Media and supplements were purchased from Life Technologies unless specified otherwise. To test the sensitivity of 152 cell lines derived from several types of solid tumours or haematological malignancies (AML, lymphoma, bladder, central nervous system, colorectal, gastric, head and neck, liver, lung, breast, melanoma, ovarian, pancreas, prostate, renal, sarcoma and uterine) to S63845, cells were grown at 37 °C in a humidified atmosphere with 5% CO in RPMI 1640 medium (25 mM HEPES, with l-glutamine, Biochrom) supplemented with 10% (v/v) FBS (Sigma) and 0.1 mg ml−1 gentamicin (Life Technologies). Different culture media were used for VCap (DMEM, 10% FCS, 1% gentamycin), CALU1 (McCoy, 10% FCS 1% gentamycin) and U87MG (EMEM, 10% FCS 1% gentamycin). These cell lines were provided by the Children’s Hospital Cologne, the University Hospital Freiburg or the NCI or were purchased from ATCC, DSMZ, JCRB, ECACC or KCBL. Two cell lines used in this study were present in the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee (ICLAC): NCI-H929 and U-937. For our study, H929 cells were obtained from authentic stocks (ATCC CRL-9068 and DSMZ ACC-163) and U937 cells were authenticated by STR analysis. All cell lines used in this study were verified to be mycoplasma negative before undertaking any experiments with them. Cell viability was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colourimetric assay. Cells were seeded in 96-well microplates at a density to maintain control (untreated) cells in an exponential phase of growth during the entire experiment. Cells were incubated with compounds for 48 h followed by incubation with 1 mg ml−1 MTT for 4 h at 37 °C. Lysis buffer (20% SDS) was added and absorbance was measured at 540 nm 18 h later. All experiments were repeated at least three times. The percentage of viable cells was calculated and averaged for each well: per cent growth = (OD treated cells/OD control cells) × 100, and the IC , the concentration where the optical density was reduced by 50%, was calculated by a linear regression performed on the linear zone of the dose–response curve. Cells were harvested from exponential phase cultures, counted and plated in 96-well flat-bottom microtitre plates at a cell density depending on the cell line’s growth rate (4,000 to 30,000 cells for solid-tumour-derived cell lines, 10,000 to 60,000 for haematological cancer-derived cell lines). After a 24-h recovery period to allow the cells to resume exponential growth, 10 μl of culture medium (four control wells per plate) or of culture medium with the test compound were added by a liquid-handling robotic system and treated or untreated cells were cultured for a further 3 days. Compounds were applied in half-log increments at 10 concentrations in duplicate. After treatment of cells, 20 μl per well CellTiter-Blue reagent (Promega) was added. After incubation for up to 4 h, fluorescence was measured by using the Enspire Multimode Reader (Perkin Elmer, excitation λ = 531 nm, emission λ = 615 nm). IC values were calculated by 4-parameter, nonlinear curve fit using Oncotest Warehouse Software. To test the activity of S63845 in combination with the kinase inhibitors trametenib, tarceva, PLX-4032 and lapatinib, SK-MEL-28, BT-474, A2058, SK-Mel-2 and PC-9 cells were seeded into 96-well plates. After 24 h, cells were treated with the indicated compounds for 72 h and assayed for viability using the CellTiter-Glo reagent (Promega). Luminescence was measured at 0, 24, 48 and 72 h on independent plates seeded and treated at the same time. Results were normalized to the samples without treatment at time 0 h. To test the sensitivity of the multiple myeloma cell lines to S63845 cells were seeded into 96-well plates at 5,000 cells per well and treated at 5-point 1:8 serial dilutions of compounds starting from 10 μM. Cell viability was assessed at 48 h using the CellTiter-Glo Assay (Promega) following the manufacturer’s instructions and the plates were read using an Envision luminescence plate reader (Perkin Elmer). Results were normalized to the viability of cells that had been treated with 0.1% DMSO (vehicle) in medium for 48 h. The IC values were calculated using nonlinear regression algorithms in Prism software (GraphPad). To test the dependence of H929 cells on BCL-2, BCL-X , BCL-W, MCL1 or A1/BFL1 for their sustained survival, pools of cells stably expressing Cas9 (mCherry+) and inducibly expressing the relevant single guide RNA (sgRNA) (GFP+) were purified by flow cytometry (BD Biosciences) and seeded into 96-well plates (5,000 cells per well) in triplicates and their viability was determined by using the CellTiter-Glo assay 72 h after the addition of doxocycline (1 μg ml−1) to induce expression of the sgRNA targeting the corresponding genes. The data were normalized to the viability of cells infected with the empty vector. In some experiments, the viability (determined by propidium iodide exclusion) of the cells with or without co-treatment with the pan-caspase inhibitor QVD-OPh (25 μM; MP Biomedicals) for 12 h was also determined. Freshly purified mononuclear cells from AML patient samples were adjusted to a concentration of 2.5 × 105 per ml1 and 100 μl of cell suspensions were aliquoted per well into 96-well plates (Sigma). Cells were then treated with S63845, cytarabine (Pfizer), ABT-199 (Active Biochem) or idarubicin (Sigma) over a 6 log concentration range from 1 nM to 10 μM for 48 h and incubated at 37 °C, 5% CO . Cells were then stained with the Sytox blue nucleic acid stain (Invitrogen) and fluorescence measured by flow cytometric analysis using a LSR-II Fortessa machine (Becton Dickinson). FACSDiva software was used for data collection, and FlowJo software for data analysis. Blast cells were gated using forward and side light scatter properties. Viable cells excluding Sytox blue were determined at six concentrations for each drug and the 50% lethal concentration (LC , in μM) was calculated using nonlinear regression algorithms in Prism software (GraphPad). NCI-H929 cells were treated with the indicated compounds for 4 h, centrifuged and washed with binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl ). Cells were incubated with 200 μl of binding buffer containing Annexin V–Alexa fluor 488 (Invitrogen) and propidium iodide (Sigma) for 15 min at 20 °C in the dark. 400 μl of binding buffer was added and samples were kept at 4 °C before flow cytometry analysis. For each sample, 104 cells were analysed by flow cytometry in an Epics XL/MCL flow cytometer (Beckman Coulter). Fluorescence was collected at 520 nm (Alexa fluor 488) and 630 nm ( propidium iodide). Human Burkitt lymphoma-derived cell lines and mouse Eμ-Myc lymphoma cell lines were plated at a density of 4 × 104 cells per well in flat-bottomed 96-well plates. These cells were treated with increasing doses of S63845 (typically 0.008, 0.025, 0.04, 0.2, 1, 5 μM) for 24 h. Cells were stained with Annexin V-FITC and propidium iodide, analysed on a FACS Calibur and live cells (Annexin V negative/propidium iodide negative) were recorded. Data are presented as per cent cell death induction relative to cells cultured in medium alone. Twenty-four hours after seeding, cells were treated with the indicated compounds for 6 h and harvested in lysis buffer (10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl , 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails (Calbiochem)). Cleared lysates (5 μg protein) were prepared for immunodetection of cleaved PARP (a marker of apoptosis) by using the MSD apoptosis panel whole cell lysate kit (MSD) in 96-well plates according to manufacturer’s instructions, and were analysed on the Sector Image 2400. NCI-H929 cells were treated with S63845 for 4 h, washed with PBS and harvested in lysis buffer delivered with the cytochrome c release apoptosis assay kit (Qiagen). Cells were then homogenized using an ice-cold tissue grinder (40 passes). Homogenates were centrifuged at 700 g for 10 min at 4 °C. The supernatants were transferred into fresh tubes and centrifuged at 10 000 g for 30 min at 4 °C. The supernatants were collected as cytosolic fractions. Cytochrome c release was determined by western blotting using the cytochrome c antibody provided in the kit. Lysates were also analysed by immunoblotting using an anti-LDH antibody (Rockland 200-1173; used as protein loading control). Total protein extracts of myeloma cells were generated in lysis buffer (20 mM Tris-HCl pH 7.4, 135 mM NaCl, 1.5 mM MgCl , 1 mM EDTA, 10% glycerol) containing 1% Triton X-100 and complete protease inhibitors (Roche). Protein extracts of the other cell lines were generated in lysis buffer containing 10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl , 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails (Calbiochem). Lysates were stored at −80 °C. Protein content was quantified using the Bradford assay (Bio-Rad). Lysates were diluted with LDS sample buffer (Invitrogen) at a 3:1 ratio and denatured at 95 °C for 7–10 min. 20–40 μg of protein extracts were separated by SDS–PAGE (NuPAGE 10% Bis Tris gels) and proteins transferred onto nitrocellulose membranes. The membranes were blocked in 5% skimmed milk in PBS and 0.1% Tween20 (blocking buffer) before incubation with antibodies. Rat monoclonal antibodies to BAX (21C10; WEHI) or BAK (7D10; WEHI) and mouse monoclonal antibody against HSP70 (N6; used as a loading control) were used. All antibodies were diluted in blocking buffer. Commercially available antibodies were also used: rabbit polyclonal antibodies against MCL1 (Santa Cruz, S-19, sc-819), PARP (Cell Signaling, 9542), BIM (Cell Signaling, C34C5 2933), Phospho-ERK (Cell Signaling, 9101), total ERK (Cell Signaling, 9102), BAK (BD 556396), BAX (Santa Cruz, sc-493) BCL-X (Transduction Laboratory, 610212) and mouse monoclonal antibodies against actin (Millipore, MAB1501R; used as a loading control), NOXA (Calbiochem, OP180), Flag-M2 (Sigma) and p53 (Santa Cruz, sc-126). HeLa cells were transiently transfected, using the Effecten reagent (Qiagen), with expression vectors encoding 3× Flag-tagged MCL1, BCL-X or BCL-2 (p3×Flag–CMV10, Sigma). After 24 h, cells were treated for 4 h with S63845 and then harvested in lysis buffer (10 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl , 1 mM EDTA, 0.4% Triton X100, protease and phosphatase inhibitors cocktails (Calbiochem)). The cleared lysates were subjected to immunoprecipitation with anti-Flag M2 agarose beads (Sigma). The immunoprecipitates and inputs were analysed by immunoblotting using the antibodies listed above. Total RNA was extracted using RNeasy mini kit with DNase I treatment (Qiagen) and reverse transcripted using a high-capacity cDNA reverse transcription kit with RNAse inhibitor (Life Technologies). Conventional real-time PCR was performed on an ABI 7900HT system in 50 μl reaction volumes containing 2× TaqMan universal PCR master mix, 2.5 μl of 20× target/control assay mix and 5 μl of respective cDNA in an optical 96-well plate. NTCs (no template controls) using RNase-free water were included in the plate. Cycling conditions were 95 °C (10 min), followed by 40 PCR cycles at 95 °C (15 s) and 60 °C (1 min). TaqMan Gene Expression Assays (Life Technologies) for MCL1 evaluated the anti-apoptotic long (L) isoform NM_021960 (reference assay Hs01050896_m1). Two out of five reference genes including GAPDH, PPIA, 18S, UBC and SDHA, were selected on geNorm software as the optimal number of reference target genes (geNorm pairwise variation cut off V < 0.15). As such, the optimal normalization factor was calculated as the geometric mean (GM) of reference targets SDHA and PPIA (ref genes) and calculation of −ΔC was achieved as follows: Data are presented as fold change of relative quantification calculated as 2–ΔΔCt, with . Pair-wise comparisons were evaluated with a t-test. Aliquots of cells were stained in 24G2 (anti-FcγR, (Fcγ gamma receptor)) antibody containing hybridoma supernatant, containing fluorescently (FITC, R-PE or APC) labelled monoclonal antibodies against cell surface markers, and analysed on an LSR11C (Becton Dickinson) excluding propidium iodide + (dead) cells. The following antibodies were used: anti-CD25 (clone PC61), anti-CD4 (clones GK1.5 (Biolegend) and H129), anti-CD8 (clones 53-6.7 (Biolegend) and YTS 169), CD44 (clone IM7), anti-B220 (220 kDa form of CD45 expressed on B cells, clones RA3-6B2 (Biolegend)), anti-GR1 (granulocyte antigen 1, clone RB6-8C5), anti-MAC1 macrophage antigen 1, clone M1/70), anti-SCA1 (stem cell antigen 1, clone E13-161.7), anti-c-KIT (clone 2B8 (Biolegend)), anti-TCR (T cell receptor, Biolegend), anti-TER119 (clone TER-119), anti-IgM (clone 5.1), anti-IgD (clone 11-26C), anti-Ly5.1 (clone A20.1) and anti-Ly5.2 (clone S.450-15.2). Data were processed using FlowJo Version 9.9 (TreeStar). Blood cell counts and cell subset composition were determined using an ADVIA 2120 haematology analyser (Siemens). The vectors for the constitutive expression of Cas9 and the inducible expression of the sgRNAs have been previously described30. To target the BCL-2 family members, sgRNAs were designed ( http://crispr.mit.edu) and cloned into pFH1tUTG (ref. 30) with the exception of sgRNAs for MCL1, which have been previously described30. The sequences of the sgRNAs used in this study as well as the primers for amplifying the targeted regions for DNA sequence analysis are detailed in Supplementary Tables 1 and 2, respectively. The vectors to express the BIM variants have been previously described12, 13. To produce lentiviruses, the constructs of interest were co-transfected into HEK293T cells with the packaging viruses pMDLg/pRRE, pRSV RRE and pCMV VSV-G (all from Addgene) using the Effectene transfection reagent (Qiagen). The lentiviruses were harvested, filtered and used to infect target cells as previously described13, 30. Multiple-myeloma-derived cell lines were serially infected with lentiviruses that stably co-express Cas9 and the fluorescent marker, mCherry, and inducibly express the indicated sgRNAs plus stably express GFP. Double positive cells (mCherry+ GFP+) were purified using a BD FACSAria Fusion Sorter (BD Biosciences). Expression of the sgRNA was induced by the addition of doxycycline (1 μg ml−1; Sigma). The experiments targeting BCL-2, BCL-X , BCL-W, MCL1 and BFL1 were undertaken with pools of infected cells. To generate the BAX−/−,BAK−/− H929 clone, a BAX-deficient H929 clone was infected with a lentivirus expressing a sgRNA to target BAK, re-cloned and verified by DNA sequencing and western blotting (Fig. 2c). Sequences of sgRNAs and primers for targeted PCR used in this study are shown in Supplementary Tables 1 and 2. DNA sequence verification was carried out as previously described30. Briefly, genomic DNA was isolated using the DNeasy kit (Qiagen) and mutation of targeted DNA confirmed by the Illumina MiSeq30. The unique PCR primers with overhang sequences for each sgRNA are listed in Supplementary Table 2. Graphpad Prism software was used for generating Kaplan–Meier animal survival plots of vehicle and S63845 treated mice and performing statistical analysis (using a log-rank test (Mantel–Cox)). Graphpad Prism was also used to perform multiple unpaired two-tailed t-tests of vehicle-treated and S63845-treated mice to look for significant changes in the data generated from the ADVIA analysis of the blood and from the FACS analysis of the number of different cells present in the spleen, thymus, lymph nodes and bone marrow. Graphpad Prism was used to generate IC curves for cell lines treated with S63845 in vitro. GraphPad Software was used for statistical analysis. All data are expressed as mean ± s.d. P < 0.05 was considered to be significant. The PDB deposition code for the X-ray structure of the MBP-MCL1 complex with S63845 is 5LOF.


News Article | February 15, 2017
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KrasG12DLSL/+, KrasG12DFSF/+, R26CreERT2 and R26Cag-LSL-Luc mice were generated by T. Jacks and obtained through the Jackson Laboratory30, 31, 32, 33. Tp53Frt/Frt mice were generated by D. Kirsch and obtained through the Jackson Laboratory34. The R26mTmG strain was generated by L. Luo and obtained through the Jackson Laboratory35. The Smarcb1Loxp/LoxP strain was provided by C. Roberts36. The Pdx1-Cre strain was obtained from A. M. Lowy through the Jackson Laboratory37. The Ptf1aCre/+ and Tp53LoxP/LoxP strains were provided by R.A.D.38, 39. The R26Cag-FlpoERT2 was generated by A. Joyner and obtained from the Jackson Laboratory40. The Cdh1Cfp strain was generated by H. Clevers and obtained through the Jackson Laboratory41. The KrasG12DFSF/+; Tp53Frt/Frt; R26CreERT2 were kept in a C57BL/6 background, the other strains were kept in a mixed C57BL/6 and 129Sv/Jae background. All animal studies and procedures were approved by the UTMDACC Institutional Animal Care and Use Committee. Animals were killed when sick or when they developed tumours larger than 15 mm in their greater diameter or ulcerated lesions. KC: KrasG12DLSL/+-Pdx1-Cre; KPC∆/∆: KrasG12DLSL/+-Tp53LoxP/LoxP-Pdx1-Cre; KSC∆/∆: KrasG12DLSL/+-Smarcb1LoxP/LoxP-Pdx1-Cre; KPSC∆/∆: KrasG12DLSL/+-Tp53LoxP/LoxP-Smarcb1LoxP/LoxP-Pdx1-Cre; CS∆/∆: Smarcb1LoxP/LoxP-Pdx1-Cre; KPC∆/∆-R26mTmG-Cdh1Cfp: KrasG12DLSL/+-Tp53LoxP/LoxP-Pdx1-Cre-R26mTmG/+-Cdh1Cfp/+; R26CreERT2/+-KPFrt/Frt: R26CreERT2/+-KrasG12DFSF/+-Tp53Frt/Frt; R26Cag-FlpoERT2/+-KP∆/∆: R26Cag-FlpoERT2/+-KrasG12DLSL/+-Tp53LoxP/LoxP; R26Cag-FlpoERT2/Cag-LSL-Luc/Cag-LSL-Luc-KP∆/∆: R26Cag-FlpoERT2/Cag-LSL-Luc/Cag-LSL-Luc-KrasG12DLSL/+-Tp53LoxP/LoxP; R26Cag-FlpoERT2/+-KPS∆/∆: R26Cag-FlpoERT2/+-KrasG12DLSL/+-Tp53LoxP/LoxP-Smarcb1LoxP/LoxP. Correct geneotype was determined by PCR analysis and gel electrophoresis at birth and at death. Males and females were equally represented in experimental cohorts. R26Cag-FlpoERT2/Cag-LSL-Luc-KP∆/∆: KrasG12DLSL and R26Cag-LSL-Luc are in cis. No sex bias was introduced during the generation of experimental cohorts. To generate pLSM5, a synthetic cassette (Geneart, Life Technologies) containing the U6 promoter and the Cre recombinase sequence under the human keratin 19 promoter (−1,114, +141) flanked by 2 TATA-Frt sites (XbaI-U6-TATA-Frt-EcoRI-hKrt19-NheI-Cre-TATA-Frt-HpaI) was cloned into the XbaI/HpaI site of the pSICO vector. A DNA fragment was liberated by XbaI/KpnI digestion and cloned into the XbaI/KpnI sites of the pLB vector42. The introduction of the TATA box into the Frt sites was designed as previously described43. To generate pLSM2, the human Keratin 19 promoter was cloned into the NotI /NheI sites of the pSICOR vector. The Flpo cassette was cloned into the NheI/EcoRI sites of the pSICOR-hKrt19 (pLSM1). A DNA fragment was liberated by KpnI/XbaI digestion and inserted into the KpnI/XbaI sites of the pLB vector to obtain the pLSM2 vector. The shRNA oligos were cloned into the HpaI/XhoI site as previously described43. All the constructs were verified by restriction digestion and sequencing. The pSICO, pSICOR, and pSICO-Flpo were made by T. Jacks31, 43. The pLB vector was created by S. Kissler. The pMSCV-LoxP-dsRed-Loxp-eGFP-Puro-WPRE vector was used for virus titration in HEK293 cells and provided by H. Clevers44. All plasmids were obtained through Addgene. The pMSCV-Neo vector was purchased from Clontech. shRNA sequences: Smarcb1-1 (5′-GGAAGAGGTGAATGATAAA-3′), Smarcb1-855 (5′-AGATAGGAACACAAGGCGAAT-3′), Smarcb1-857 (5′-GCCATCCGAAATACCGGAGAT-3′), Atf2 (5′-GAAGTTTCTAGAACGAAATAG-3′), c-Jun (5′-CAGTAACCCTAAGATCCTAAA-3′), Kras (5′-GGAAACAAGTAGTAATTGA-3′), Ern1 (5′-GCTGAACTACTTGAGGAATTA-3′), Mkk4 (5′-CCCATACATTGTTCAGTTCTA-3′), negative control (5′-GCAAGCTGACCCTGAAGTTCAT-3′). To amplify integrated vector from genomic DNA the following oligonucleotides were used: forward, 5′-CCCGGTTAATTTGCATATAATATTTC-3′; reverse, 5′-CATGATACAAAGGCATTAAAGCAG-3′. For constitutive knock-down experiments, the pLKO.1 system was used. Cells were briefly selected in puromycin before experiments. The murine Myc open reading frame was purchased from Genecopoeia and subcloned into the EcoRI/BglII sites of the pMSCV-Neo vector. The pLenti-PKG Gfp-Puro was obtained from Addgene45. In the pLSM2-shRNA system/mouse strain, we crossed a latent allele of oncogenic KrasG12DFSF/+ that can be activated by Flpo-mediated recombination with a conditional Tp53Frt/Frt allele that, similarly, can be ablated in a time-restricted, tissue-specific manner by expressing a codon-optimized Flpo recombinase (provided by lentiviral delivery and under a tissue specific promoter)31, 34, 46. In addition, we introduced a tamoxifen-inducible Cre recombinase (CreERT2) that is expressed in virtually all tissues30. The pLSM2-shRNA system/vector was designed as follows. The lentiviral vector expresses the codon-optimized Flpo recombinase under the human KRT19 promoter and a constitutive shRNA under the U6 promoter. The entire cassette is flanked by LoxP sites and can be removed by Cre-mediated recombination in a time-restricted manner. The orthotopic injection of the virus results in the activation of oncogenic Kras and inactivation of Tp53 along with the RNAi-mediated depletion of Smarcb1 in the pancreatic epithelial compartment. The treatment with caerulein (performed according to the staggered protocol described previously6), starts 1 week after the viral injection and results in robust activation of a ductal differentiation program in the acinar compartment (acinar ductal metaplasia) and in a proliferative response6. Tamoxifen treatment results in Cre-mediated recombination at the LoxP sites in the genome of the integrated provirus, deletion of the shRNA cassette and restoration of expression of the gene target. In the pLSM5-shRNA system/mouse strain, we crossed mouse strains carrying a latent oncogenic KrasG12DLSL/+ allele (activated by Cre-mediated recombination) with a conditional Tp53LoxP/LoxP allele (along with a conditional Smarcb1LoxP/LoxP allele in some experiments) that, similarly, can be ablated in a time-restricted, tissue-specific manner by expressing a Cre recombinase (provided by lentiviral delivery)32, 36, 38. In addition, we introduced a tamoxifen-inducible Flpo recombinase (FlpoERT2) which is expressed in virtually all tissues under a strong promoter (CAG)40. The pLSM5-shRNA lentiviral vector expresses a codon-optimized Cre recombinase under the human KRT19 promoter and a latent shRNA that can be activated by Flpo-mediated recombination and the deletion of a Frt-Stop-Frt cassette. A TATA-box cassette was introduced into the Frt sites to increase shRNA expression upon Flpo-mediated recombination. The system allows the generation of primary tumours and the depletion of a gene of interest at a desired time. Infectious viral particles were produced using psPAX2 and pMD2G helper plasmids. For transfection, 293T cells were cultured in DMEM containing 10% FBS (Gibco), 100 IU ml−1 penicillin (Gibco), 100 μg ml−1 streptomycin (Gibco) and 4 mM caffeine (Sigma-Aldrich) and transfected using the polyethylenimine method. Virus-containing supernatant was collected 48–72 h after transfection, spun at 3,000 r.p.m. for 10 min and filtered through 0.45-μm low-protein-binding filters (Corning)47. High-titre preparations were obtained by multiple rounds of ultracentrifugation at 23,000 r.p.m. for 2 h each. Viral titre was quantified in HEK293T cells stably transduced with a Cre-inducible GFP reporter44. For orthotopic injections, a previously described protocol was partially modified13. In brief, virus was resuspended in a solution of OPTI-MEM and polybrene (8 μg ml−1). Mice were anaesthetized using a ketamine/xylazine solution (150 mg kg−1 and 10 mg kg−1, respectively). Shaved skin was disinfected with betadine and ethanol and 1-cm incisions were performed through the skin/subcutaneous and muscular/peritoneal layers. The spleen and tail of the pancreas were identified and exposed and multiple injections were performed in the pancreatic tail and body (2 × 108–5 × 108 IU per mouse). The muscular/peritoneal planes were closed using continuous absorbable sutures. The skin/subcutaneous planes were closed using interrupted absorbable sutures. Analgesia was achieved with buprenorphine (0.1 mg kg-1 BID). At 7 days after surgery, mice were treated with caerulein as previously described6. Mice were monitored for tumour formation twice per week. For tamoxifen treatment, after tumours were detected, mice were treated with tamoxifen (Sigma) by intraperitoneal injection. A total of 100 μl of tamoxifen solution (15 mg ml−1 in corn oil) was injected every other day, giving five injections in total. Treatment cycles were repeated every 2 weeks if appropriate. In orthotopic secondary transplantation studies, tamoxifen treatment was started 5 days after surgery. For orthotopic transplantations experiments, 2 × 105 cells were resuspended in a 2:1 solution of OPTI-MEM (Gibco) and Matrigel (BD Biosciences, 356231) and transplanted into the tail of the pancreas of 6–9-week-old mice in a single injection (25 μl). For subcutaneous transplantation studies, tumour cells were resuspended in OPTI-MEM (Gibco) and Matrigel (BD Biosciences, 356231) (2:1 dilution) and injected subcutaneously into the flank of 6–9-week-old NCr Nude female mice (Taconic). Liver-seeding experiments were performed as described previously48. Liver weight was measured fresh at necropsy. For transplantation in a limiting dilution, 1 × 103, 2 × 102 or 2 × 10 tumour cells were resuspended in a 2:1 solution of OPTI-MEM (Gibco) and Matrigel (BD Biosciences, 356231) and injected into the flank of 6–9-week-old NCr Nude female mice (Taconic). Mice were observed for 24–34 weeks. The TIC frequency was calculated using L-Calc Limiting Dilutions Software (Stem Cell Technologies) and expressed as proportion of TIC ± s.e.m. The following primary antibodies were used for immunofluorescence, immunohistochemistry and immunoblotting: phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (D13.14.4E, Cell Signaling Technologies #4370); phospho-MEK1/2 (Ser221, 166F8) (Cell Signaling Technologies #2338), SMARCB1 (Sigma Aldrich # HPA018248); SMARCB1 (BD Transduction Laboratories #612111); Vinculin (E1E9V, Cell Signaling Technologies #13901); vimentin (D21H3, Cell Signaling Technologies #5741); CDH1 (4A2, Cell Signaling Technologies #14472); nestin (rat-401 Millipore #Mab 353); Ki67 (Thermo Scientific #RM9106); Sox9 (Millipore #AB-5535); Pdx1 (Millipore # 06-1385); cleaved caspase 3 (A175, Cell Signaling Technologies #9661); phospho-p38 (D3F9) (Cell Signaling Technologies #4511); p38α (Cell Signaling Technologies #9218); JNK (Cell Signaling Technologies #9252); phospho-JNK (Thr183/Tyr185, 81E11, Cell Signaling Technologies #4668); ATF2 (20F1 Cell Signaling Technologies #9226), phospho-ATF2 (Thr69/71, Cell Signaling Technologies #9225); c-Jun (60A8, Cell Signaling Technologies #9165); phospho-c-Jun (Ser73, D47G9, Cell Signaling Technologies #3270); ubiquitin (Cell Signaling Technologies #3933); IRE1-α (14C10, Cell Signaling Technologies #3294); PERK (D11A8, Cell Signaling Technologies #5683); XBP-1 s (D2C1F, Cell signaling Technologies #12782); ATF6 (70B1413, Abcam #11909); ATF6 (NovusBio # NBP1-77251); SEK1/MKK4 (Cell Signaling Technologies #9152); phospho-SEK1/MKK4 (Ser257, C36C11, Cell Signaling Technologies #4514); c-Myc (D3N8F, Cell Signaling Technologies #13987). The following chemical reagents were used: gemcitabine (LC Laboratories), bortezomib (LC Laboratories), carfilzomib (LC Laboratories), NVP-AUY-922 (LC Laboratories), ganetespib (Selleck Chemicals) SP600125 (LC Labs), BIRB796 (LC Labs), tunicamycin (Selleck Chemicals). Senescence-associated β-galactosidase staining was performed with a senescence β-Galactosidase Staining Kit (Cell Signaling Technologies) according to the manufacturer’s instructions. Tumour-derived cells and primary lines were cultured in vitro for <5 passages prior to experimentation. Aldefluor-based cell sorting (Stem Cell Technologies) was performed according to the manufacturer’s instructions. Cells showing a fluorescence signal above the average of the diethylaminobenzaldehyde-treated negative controls were considered positive. Protein synthetic rate was assessed using the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Life Technologies) according to the manufacturer’s instructions. The rate of incorporation of OPP was assessed by FACS analysis. Cells cultured in the presence of 20 μM Cycloheximide (Sigma Aldrich) were used as negative technical controls. After staining, samples were acquired using a BD FACS Canto II flow cytometer. Cell sorting experiments were performed using BD Influx cell sorter. For details see ref. 49. Data were analysed by FlowJo (Tree Star). Patient-derived samples were obtained from patients who had given informed consent under Institutional Review Board (IRB)-approved protocols LAB07-0854 chaired by J.B.F. (UTMDACC) and IRB00044588 chaired by L. D. Wood (JHMI). The establishment of human PDX lines was described in detail previously50, 51. Passage-1 PDXs were dissociated using collagenase and dispase (collagenase IV–dispase 4 mg ml−1; Invitrogen) at 37 °C for 1 h and single-cell suspensions were then transduced with a lentiviral GFP reporter (pLenti-PKG GFP-Puro) and transplanted into NOD SCID immunocompromised mice. Experimental cohorts were generated by serial transplantations in vivo. Cells were isolated from primary pancreatic tumours as previously described52. Cells derived from primary mouse tumours were kept in culture as spheres in semi-solid media for <5 passages. After explant, tumours were digested at 37 °C for 1 h (collagenase IV-dispase 4 mg ml−1; Invitrogen). Single-cell suspensions were plated in DMEM (Lonza) supplemented with 2 mM glutamine (Invitrogen), 10% FBS (Lonza), 40 ng ml−1 hEGF (PeproTech), 20 ng ml−1 hFGF (PeproTech), 5 μg ml−1 h-insulin (Roche), 0.5 μM hydrocortisone (Sigma), 100 μM β-mercaptoethanol (Sigma), 4 μg ml−1 heparin (Sigma), penicillin (Gibco) 100 IU ml−1 and streptomycin (Gibco) 100 μg ml−1. Methocult M3134 (StemCell Technologies) was added to the culture medium to a final concentration of 0.8% (v/v)) to keep tumour cells growing as clonal spheres and not aggregates. Spheres were collected and digested with 0.25% trypsin (Gibco) to single cells and re-plated. For 2D tumour cultures, cells were kept in DMEM containing 10% FBS (Gibco), 100 IU ml−1, penicillin (Gibco) and 100 μg ml−1 streptomycin (Gibco). For in vivo transplantation studies, low-passage (<5) tumour cells were used. For GDAs, single-cell suspensions were generated using collagenase and dispase (collagenase IV–dispase 4 mg ml−1, Invitrogen) and transplanted immediately into recipient mice. Experimental cohorts were generated by serial transplantations in vivo. The following was performed as previously described53 with modifications. Pancreata were harvested and digested at 37 °C for 45 min (collagenase IV, 4 mg ml−1) and passed through a 100-μm nylon cell strainer to separate the acinar fraction from larger ducts. The ductal fraction underwent additional digestion with 0.25% trypsin (Gibco) for 5 min at 37 °C and mechanical disruption. The two fractions were combined and plated on collagen IV-coated plates (Corning) in modified PDEC medium: DMEM/F12 (Gibco), 15 mM HEPES (Invitrogen), 5 mg ml−1 d-glucose (Sigma Aldrich), 1.22 mg ml−1 nicotinamide (Sigma Aldrich), 5 nM 3,3,5-tri-iodo-l-thyronine (Sigma Aldrich), 1 μM dexamethasone (Sigma Aldrich), 100 ng ml−1 cholera toxin (Sigma Aldrich), 5 ml l−1 insulin-transferrin-selenium (BD), penicillin (Gibco), 100 μg ml−1 streptomycin (Gibco), 0.1 mg ml−1 soybean trypsin inhibitor (Sigma Aldrich), 40 ng ml−1 EGF (Sigma Aldrich), 25 μg ml−1 bovine pituitary extract (Invitrogen), 100 μM β-mercaptoethanol (Sigma) and 10% FBS (Gibco). Cells were passaged at low confluency until exhaustion or escaper clones were established. For drug treatments, spheres were collected, washed, digested with trypsin and repeatedly counted (Countless, Invitrogen). Equal numbers of live cells were incubated with bortezomib (5 nM), carfilzomib (5 nM), NVP-AUY-922 (50 nM), ganetespib (50 nM), 4-hydroxy-tamoxifen (250 nM) and tunicamycin (200 nM). Spheroids were manually counted under a Nikon Eclipse Ti microscope using a click-counter. Experiments were repeated at least three times and error bars represent the s.d. of technical replicates from a representative experiment. For orthotopic end point survival studies, 6–9-week-old female mice were transplanted orthotopically with 2 × 105 cells resuspended in a 2:1 solution of OPTI-MEM (Gibco) and Matrigel (BD Biosciences, 356231). GEMM-derived-allografts and PDXs were briefly dissociated and passaged in vivo in NCr Nude and NOD SCID female mice, respectively, to limit the phenotypic changes associated with 2D cultures. Tumour volumes were measured according to the formula l × w2/2, where w represents tumour width. Clinical response was determined as the ratio of tumour volume at the end of the treatment to the volume at the beginning of the treatment. Gemcitabine was administered intraperitoneally at 100 mg kg−1 every 4 days for 16 days; NVP-AUY-922 was administered intraperitoneally at 75 mg kg−1 every other day for 16 days; BIRB796 was administered by oral gavage every second day at 40 mg kg−1 for 16 days; SP600125 was administered intraperitoneally at 40 mg kg−1 every day for 16 days. Gemcitabine was dissolved in phosphate buffer saline, AUY922 was dissolved 10% DMSO/25% water/65% PEG 400, SP600125 was resuspended in PBS and DMSO and BIRB796 was prepared as previously reported54. Animals were imaged on a 4.7T Bruker Biospec (Bruker BioSpin) equipped with 6-cm inner-diameter gradients and a 35-mm inner-diameter volume coil. Multi-slice T2-weighted images were acquired in coronal and axial geometries using a rapid acquisition with relaxation enhancement (RARE) sequence with TR/TE of 2,000/38 ms, matrix size 256 × 192, 0.75-mm slice thickness, 0.25-mm slice gap, 4 × 3-cm FOV, 101-kHz bandwidth, 3 NEX. Axial scan sequences were gated to reduce respiratory motion. Detection of luciferase activity was performed in an IVIS-100 imaging system. Five minutes before the procedure, mice were injected intraperitoneally with d-luciferin, bioluminescence substrate (Perkin Elmer) according to the manufacturer’s instructions. Living Image 4.3 software (Perkin Elmer) was used for analysis of the images after acquisition. Tumour samples were fixed in 4% formaldehyde for 24 h at room temperature, moved into 70% ethanol for 12 h, and then embedded in paraffin (Leica ASP300S). After cutting (Leica RM2235) and baking at 60 °C for 20 min for de-paraffinization, slides were treated with Citra-Plus Solution (BioGenex) for antigen unmasking according to the manufacturer’s instructions. For immunohistochemical staining, endogenous peroxidases were inactivated by 3% hydrogen peroxide at room temperature for 15 min. Non-specific signals were blocked using 5% BSA and 5% goat serum for 1 h. Tumour samples were stained with primary antibodies for 12 h at 4 °C and the Mouse on Mouse Kit (Vector Laboratory) was used when appropriate according to the manufacturer’s instructions. For immunostaining, ImmPress (Vector Laboratory) were used as secondary antibodies and Nova RED (Vector Laboratory) was used for detection. Images were captured with a Nikon DS-Fi1 digital camera using a wide-field Nikon Eclipse Ci microscope. For immunofluorescence, secondary antibodies conjugated to Alexa488, Alexa647 and Alexa555 (Molecular Probes) were used. Images were captured with a Hamamatsu C11440 digital camera, using a wide-field Nikon EclipseNi microscope and a Nikon high-speed multi-photon confocal microscope A1 R MP. Total staining score was weighed to the intensity and prevalence (percentage of positive tumour cells and intensity score of 0 to 3) in random fields at 20× magnification. Quantitative analysis was performed using Image J and Immunoratio programs according to the providers’ instructions at 20× original magnification. TEM was performed at the UTMDACC High Resolution Electron Microscopy Facility. Samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h. After fixation, the samples were washed and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, post-fixed with 1% buffered osmium tetroxide for 30 min, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated and embedded in LX-112 medium. The samples were polymerized at 60 °C for 2 days. Ultra-thin sections were cut using a Leica Ultracut microtome, stained with uranyl acetate and lead citrate in a Leica EM Stainer and examined using a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV. Digital images were obtained using an AMT Imaging System (Advanced Microscopy Techniques Corp). Protein lysates were resolved on 5–15% gradient polyacrylamide SDS gels (Bio-Rad) and transferred onto PVDF membranes (Bio-Rad) according to the manufacturer’s instructions. Membranes were incubated with the indicated primary antibodies, washed in TBST buffer and probed with HRP-conjugated secondary antibodies. The detection of bands was carried out upon chemi-luminescence reaction followed by film exposure (Denville Scientific). In vitro and in vivo data are presented as the mean ± s.d. Results from limiting dilutions analysis (LDA) were expressed as the proportion of TIC ± s.e.m. Differences in stem-cell frequencies between groups were determined using a chi-squared test (2-tailed)55, 56. Comparisons between biological replicates were performed using a two-tailed Student’s t-test. Results from survival and incidence experiments were analysed with a log-rank (Mantel–Cox) test and expressed as Kaplan–Meier survival curves. Results from contingency tables were analysed using the two-tailed Fisher’s exact test (GraphPad software). Group size was determined on the basis of the results of preliminary experiments. No statistical methods were used to determine sample size. Group allocation and analysis of outcome were not performed in a blinded manner. Samples that did not meet proper experimental conditions were excluded from the analysis. DNA and RNA were isolated using DNeasy Blood and Tissue Kit (Qiagen) and RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Gene expression profiling was performed at the UTMDACC Microarray Core Facility on a Gene Chip Mouse Genome 430 2.0 Array (Affymetrix). The robust multi-array average method was used with default options (with background correction, quantile normalization, and log transformation) to normalize raw data from batches using R/Bioconductor’s affy package (12925520) and analysed with GSEA c3.tft.v4.0 (TFT) and c6.all.v4.0. (Oncogenic Signatures); HOMER (20513432) was also used to identify significantly enriched biological pathways or processes for the differentially expressed genes57, 58. Subgroup information (Classical, QM-PDA, Exocrine-like) for each gene was provided to a heuristic optimization method (stochastic gradient descent) to minimize objective function. The objective function output was used to calculate decision boundaries with a support vector machine approach to optimize the partitioning of subtypes. The obtained microarray signal values for each probe were used for proper classification. The decision surface for multi-class datasets was plotted with Python package matplotlib. To control for random occurrence, we permutated the classification subtypes provided to the stochastic gradient descent function and randomized trainings yield ambiguous classifications, suggesting that gene expression signatures in our model is overlapping with the previous pancreas cancer subgroups. Clinical and pathological data for patient samples are provided in Supplementary Table 1. Microarray data supporting the findings of this study have been deposited in the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE83754. All other data are available from the corresponding author (G.G.) upon reasonable request.


News Article | February 15, 2017
Site: www.nature.com

All mice were females from a mixed background, housed under standard laboratory conditions, and received food and water ad libitum. All experiments were performed in accordance with the guidelines of the Animal Welfare Committee of the Royal Netherlands Academy of Arts and Sciences, The Netherlands. R26-Confetti;R26-CreERT2, R26-TdTomato;R26-CreERT2, and R26-mTmG;R26-CreERT2 mice were injected intraperitoneally with tamoxifen (Sigma Aldrich), diluted in sunflower oil, to activate Cre recombinase at 3 weeks of age. To achieve clonal density labelling (<1 MaSC per duct), R26-Confetti mice were injected with 0.2 mg tamoxifen per 25 g body weight. To label multiple MaSCs per TEB, R26-Confetti mice were injected with 1.5 mg/25 g at 3 weeks. The clonal dose for R26-mTmG and R26-TdTomato reporter mice was 0.2 mg tamoxifen per 25 g body weight and 0.05 mg/25 g tamoxifen, respectively. Lineage-traced mice were euthanized at mid-puberty (5 weeks of age) or at the end of puberty (8 weeks of age) and mammary glands were collected. Experiments were not randomized, sample size was not determined a priori, and investigators were not blinded to experimental conditions except where indicated. Imaging of whole-mount mammary glands was performed using a Leica TCS SP5 confocal microscope, equipped with a 405 nm laser, an argon laser, a DPSS 561 nm laser and a HeNe 633 nm laser. Different fluorophores were excited as follows: DAPI at 405 nm, CFP at 458 nm, GFP at 488 nm, YFP at 514 nm, RFP at 561 nm and Alexa-647 at 633 nm. DAPI was collected at 440–470 nm, CFP at 470–485 nm, GFP at 495–510 nm, YFP at 540–570 nm, RFP at 610–640 nm and Alexa-647 at 650–700 nm. All images were acquired with a 20× (HCX IRAPO N.A. 0.70 WD 0.5 mm) dry objective using a Z-step size of 5 μm (total Z-stack around 200 μm). All pictures were processed using ImageJ software (https://imagej.nih.gov/ij/). Quantitative analysis of the whole-gland reconstructions induced with 0.2 mg tamoxifen per 25 g body weight was performed on 36 glands from nine mice at 8 weeks of age. We counted 11 luminal and 8 basal clones in subtrees starting from level 6. Quantitative analysis of the whole-gland reconstructions induced with 1.5 mg tamoxifen per 25 g body weight was performed for 10 glands from five mice at 8 weeks of age, 5 glands from three mice at 5 weeks of age, and 3 glands from two mice at 8 weeks of age (traced from 5 weeks of age). Clonal analysis and modelling were based on 606 subclones (157 basal subclones and 449 luminal subclones) from four glands from mice at 8 weeks of age. Data were collected at random and all glands induced at a clonal level were included. Subclones were defined as the density of epithelial cells of the same type (basal or luminal) and confetti colour in a given branch of a given level. Although our theoretical description models the distribution of the number of MaSCs in a TEB of level l, as we cannot access this quantity directly experimentally, we use as a proxy the density of labelled cells of a given type and confetti colour in the corresponding branch of level l (that is, the branch that was formed by the TEB considered in the model). Taking the density instead of the absolute number of labelled cells allowed us to correct for the stochastic variation of branch length that we observed (Extended Data Fig. 1e). The fourth and fifth mammary glands were dissected and incubated in a mixture of collagenase I (1 mg/ml, Roche Diagnostics) and hyaluronidase (50 μg/ml, Sigma Aldrich) at 37 °C for optical clearance, fixed in periodate–lysine–paraformaldehyde (PLP) buffer (1% paraformaldehyde (PFA; Electron Microscopy Science), 0.01M sodium periodate, 0.075 M l-lysine and 0.0375 M P-buffer (0.081 M Na HPO and 0.019M NaH PO ; pH 7.4) for 2 h at room temperature, and incubated for 2 h in blocking buffer containing 1% bovine serum albumin (Roche Diagnostics), 5% normal goat serum (Monosan) and 0.8% Triton X-100 (Sigma-Aldrich) in PBS. Primary antibodies were diluted in blocking buffer and incubated overnight at room temperature. Secondary antibodies diluted in blocking buffer were incubated for at least 4 h. Nuclei were stained with DAPI (0.1 μg/ml; Sigma-Aldrich) in PBS. Glands were washed with PBS and mounted on a microscopy slide with Vectashield hard set (H-1400, Vector Laboratories). Primary antibodies: anti-K14 (rabbit, Covance, PRB155P, 1:700) or anti-E-cadherin (rat, eBioscience, 14-3249-82, 1:700). Secondary antibodies: goat anti-rabbit or goat anti-rat, both conjugated to Alexa-647 (Life Technologies, A21245 and A21247 respectively, 1:400). For 5-ethynyl-2-deoxyuridine (EdU) cell-proliferation staining of whole-mount mammary glands, a click-it stain (Click-iT EdU, Invitrogen) was performed according to the manufacturer’s instructions before staining with primary antibodies as described above. Kidneys were dissected from embryos at embryonic day 16, fixed in PLP buffer overnight at 4 °C, and incubated for 4 h in blocking buffer containing 1% bovine serum albumin (Roche Diagnostics), 5% normal goat serum (Monosan) and 0.8% Triton X-100 (Sigma-Aldrich) in PBS. Primary antibodies were diluted in blocking buffer and incubated overnight at room temperature. Secondary antibodies diluted in blocking buffer were incubated for at least 6 h. Nuclei were stained with DAPI (0.1 μg/ml; Sigma-Aldrich) in PBS. 3.5-, 5- or 8-week-old mice (n = 3 mice for each age) were injected intraperitoneally with 0.5 mg EdU (Invitrogen) diluted in PBS. For the EdU pulse-chase experiments, EdU (0.5 mg) was injected intraperitoneally in 4-week-old mice (n = 3). Mice were euthanized 4 or 72 h after EdU injection and the fourth and fifth mammary glands were collected and processed as whole-mount glands. For analysis, 3D tile-scan images of the whole-mount glands were taken and the number of EdU+ TEBs was scored. For the quantification of the fraction of EdU+ cells, 10 EdU+ TEBs per time point were selected in a blinded manner and the number of EdU+ cells was counted manually for each selected TEB. For the pulse-chase experiment, 3 EdU+ TEBs per mouse were selected in a blinded manner and the intensity of 10 randomly picked EdU+ cells was measured for both the tip and the border area of the selected TEB and a two-tailed t-test was performed. Normal distribution was confirmed using a d’Agostino and Pearson omnibus normality test. The variance between the groups was tested with an F-test and was found to be not significantly different. Three-dimensional tile-scan images of whole-mount mammary glands and embryonic kidneys were used to manually reconstruct the ductal network by outlining the ducts. Labelled confetti cells were annotated in the schematic outline of the mammary tree, including information on the confetti colour for the mammary glands (GFP, green; YFP, yellow; RPF, red; and CFP, cyan). Using custom-made. NET software (available upon request from J.v.R.), the length and width of all the ducts, the coordinates of the branch points, and the position of the labelled cells in ducts and in TEBs were scored in these schematic outline images, which was used as input for a schematic representation of the lineage tree. Custom-made Python software (available upon request from E.H.) and the ETE2 python toolkit were used for the conversion and visualization of the schematic mammary gland lineage tree, including linkage between branches, their respective length and number of cells of each confetti colour. To depict the topology of the resulting tree, the Newick format was used to represent hierarchical structures using nested parentheses to encode information about the linkage between branches, their respective length and number of cells of each confetti colour. The origin of the gland was always located at the top of the reconstruction. R26-CreERT2;R26-mTmG mice were intraperitoneally injected with 0.2 mg tamoxifen per 25 g body weight diluted in sunflower oil (Sigma) at 3 weeks of age. At 5 weeks of age, a mammary window was inserted near the fourth and fifth mammary glands (for details, see ref. 31). Mice were anaesthetized using isoflurane (1.5% isoflurane/medical air mixture) and placed in a facemask with a custom designed imaging box. Mice were intraperitoneally injected with AZD-7762 (0.5 mg in PBS, Sigma) every 5 h during the time-lapse imaging. Imaging was performed on an inverted Leica SP8 multiphoton microscope with a chameleon Vision-S (Coherent Inc.), equipped with four HyD detectors: HyD1 (<455 nm), HyD2 (455–490 nm), HyD3 (500–550 nm) and HyD4 (560–650 nm). Different wavelengths between 700 nm and 1,150 nm were used for excitation; collagen (second harmonic generation) was excited with a wavelength of 860 nm and detected in HyD1. GFP and Tomato were excited with a wavelength of 960 nm and detected in HyD3 and HyD4. TEBs and ducts were imaged every 20–30 min using a Z-step size of 3 μm over a minimum period of 8 h. All images were in 12 bit and acquired with a 25× (HCX IRAPO N.A. 0.95 WD 2.5 mm) water objective. Single TEBs and ducts were isolated from 5-week-old MMTV-Cre;R26-loxP-stop-loxP-YFP mice. YFP expression was used to determine the localization and structure of the mammary gland. Single TEBs and pieces of ducts were micro-dissected from the fourth and fifth mammary glands. Single TEBs and ducts were digested in DMEM/F12 (GIBCO, Invitrogen Life Technologies) supplemented with hyaluronidase (300 μg/ml, Sigma Aldrich), and collagenase I (2 mg/ml, Roche Diagnostics) at 37 °C, followed by centrifugation at 550g for 10 min. The fatty layer on top and the aqueous layer in the middle were aspirated, and the remaining pellet was dissolved in 5 mM EDTA/PBS with 5% fetal bovine serum (Sigma) and kept on ice for 10 min before labelling with the following antibodies: anti-mouse CD45-Pacific blue (clone 30-F11, Biolegend), anti-mouse CD31-Pacific blue (clone 390, Biolegend), and rat anti-mouse CD140a-Pacific blue (Clone APA5, BD Bioscience). Cells were incubated for 30 min on ice in the dark, washed once in 5 mM EDTA/PBS with 5% fetal bovine serum (Sigma), and centrifuged at 250g for 5 min. Pellet was dissolved in 5 mM EDTA/PBS with 7-AAD, and sorted on a FACS AriaII Special Ordered Reseach Product (BD Biosciences). A broad FSC SSC gate was followed by a gate excluding doublets. Next, 7-AAD negative cells were gated, and from this population Lin− (CD45−, CD31−, CD140a−) and YFP+ cells were sorted into 384-well plates containing 5 μl of mineral oil that contained a 200-nl droplet of primers, dNTPs and synthetic mRNA molecules (ERCC). Single-cell mRNA sequencing was performed as described previously32. In brief, cells were sorted into 5 μl of mineral oil containing a 200-nl droplet of primers, dNTPs and synthetic mRNA molecules (ERCC). Cells were fused with this droplet by centrifugation and lysed at 65 °C, followed by room temperature and second-strand synthesis aided by a Nanodrop II liquid handling platform. The resulting cDNA was processed following the CEL-Seq2 protocol33. Libraries were sequenced on an Illumina NextSeq with 75-bp paired-end reads. The 5′ mate was used to identify cells and libraries while the 3′ mate was aligned to the mm10 RefSeq mouse transcriptome using BWA34. Analysis was performed using StemID (for details of the methodology, see ref. 26). Endothelial cells, erythrocytes and lymphocytes were filtered from the data based on expression of Cd36 (5 unique transcripts), Beta-s (1,000 unique transcripts) and Cd74 (2 unique transcripts)/Cd52 (1 unique transcript), respectively (27 cells in total). The remaining cells were normalized by down sampling to 3,000 transcripts, after which StemID26 was used for clustering and cell type annotation. Cells with fewer than 3,000 unique transcripts were discarded. In total, 91 cells were included, of which we could assign 36 cells to the luminal lineage (cluster 1 contained 9 cells, cluster 2 contained 17 cells, cluster 3 contained 2 cells and cluster 5 contained 8 cells), 51 cells to the basal lineage (cluster 5 contained 2 cells, cluster 6 contained 14 cells, cluster 7 contained 10 cells, cluster 8 contained 8 cells and cluster 9 contained 17 cells), and 4 cells to a non-epithelial origin (cluster 4). After identifying luminal and basal cell clusters, three cells that were erroneously annotated as basal cells belonging to cluster 8 were manually annotated to belong to luminal cluster 2, based on luminal and basal markers such as K8/K18 and K5/Acta2, respectively. Cluster 5 expressed higher levels of the pre-ribosomal 45S RNA, which is often found in cells of low quality. We therefore chose to omit cluster 5 from further analysis. Differential gene expression between clusters was based on a previous method35 and performed as described previously32. All data analysis with StemID and custom scripts was performed with Rstudio, version 0.99.491. The sequencing data discussed in this publication have been deposited in the Gene Expression Omnibus, and are accessible through GEO Series accession number GSE85875. Source data are available for Figs 1c–f, 2f, 3b, c, e, 4a–c and Extended Data Figs 1e–k, 2c, e–h, 3b–g, 4e, f, 6d, 8f. All other data are available from the author upon reasonable request. Custom-made. NET software to score the length and width of all the ducts, the coordinates of the branch points and the position of the labelled cells in ducts and in TEBs used as input for a schematic representation of the lineage tree is available upon request from J.v.R. Custom-made Python software and the ETE2 Python toolkit used for the conversion and visualization of the schematic mammary gland lineage tree are available upon request from E.H.


News Article | December 14, 2016
Site: www.nature.com

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Human skin tissue was obtained from healthy donors undergoing corrective breast or abdominal surgery after informed consent in accordance with our institutional guidelines. This study was approved by the Medical Ethics Review Committee of the Academic Medical Center. Split-skin grafts of 0.3 mm in thickness were obtained using a dermatome (Zimmer). After incubation with Dispase II (1 U ml−1, Roche Diagnostics), epidermal sheets were separated from the dermis and cultured in in Iscoves Modified Dulbeccos’s Medium (IMDM, Thermo Fischer Scientific) supplemented with 10% FCS, gentamycine (20 μg ml−1, Centrafarm), pencilline/streptomycin (10 U ml−1 and 10 μg ml−1, respectively; Invitrogen). Further LC purification was performed using a Ficoll gradient (Axis-shield) and CD1a microbeads (Miltenyl Biotec) as described before4, 10. Isolated LCs were routinely 90% pure and expressed high levels of Langerin and CD1a. MUTZ-LCs were differentiated from CD34+ human AML cell line MUTZ3 progenitors in the presence of GM-CSF (100 ng ml−1, Invitrogen), TGF-β (10 ng ml−1, R&D) and TNF-α (2.5 ng ml−1, R&D) and cultured as described before14. Immature DCs were differentiated from monocytes, isolated from buffy coats of healthy volunteer blood donors (Sanquin, The Netherlands), in the presence of IL-4 (500 U ml−1, Invitrogen) and GM-CSF (800 U ml−1, Invitrogen) and used at day 6 or 7 as previously described20. CD4+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) activated with phytohaemagglutinin (1 mg ml−1; L2769, Sigma Aldrich) for 3 days, enriched for CD4+ T cells by negative selection using MACS beads (130-096-533, Miltenyi) and cultured overnight with IL-2 (20 U ml−1; 130-097-745, Miltenyi) as described before5. The following inhibitors were used: rapamycin (mTOR inhibitor, tlrl-rap, Invivogen), bafilomycin A1 (V-ATPase inhibitor; tlrl-baf1; Invivogen) and MG-132 (proteasome inhibitor; 474790; Calbiochem). All cell lines were obtained from ATCC and tested negative for mycoplasma contamination, determined in 3-day-old cell cultures by PCR. Langerin and Langerin mutant W264R expression plasmid pcDNA3.1 were obtained from Life Technologies and subcloned into lentiviral construct pWPXLd (Addgene). HIV-1-based lentiviruses were produced by co-transfection of 293T cells with the lentiviral vector construct, the packaging construct (psPAX2, Addgene) and vesicular stomatitis virus glycoprotein envelope (pMD2.G, Addgene) as described previously31. U87 cell lines stably expressing CD4 and wild-type CCR5 co-receptor (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: U87 CD4+CCR5+ cells from H. K. Deng and D. R. Littman32) were transduced with HIV-1-based lentiviruses expressing sequences coding human TRIM5α33, rhesus TRIM5α33, wild-type Langerin or Langerin(W264R). NL4.3, NL4.3-BaL, SF162, NL4.3eGFP-BaL, NL4.3-BlaM-Vpr and VSV-G-pseudotyped NL4.3(ΔEnv) HIV-1 were generated as described10. All produced viruses were quantified by p24 ELISA (Perkin Elmer Life Sciences) and titrated using the indicator cells TZM-Bl. Primary LCs and MUTZ-LCs were infected with a multiplicity of infection of 0.2–0.4 and HIV-1 infection was assessed by flow cytometry at day 7 after infection by intracellular p24 staining. Double staining with CD1a (LCs marker; HI149-APC; BD Pharmigen) and p24 (KC57-RD1-PE; Beckman Coulter) was used to discriminate the percentage of CD1a+p24+ infected LCs. CD4+CCR5+ U87 parental or transduced cells were infected at a multiplicity of infection of 0.1–0.2 and HIV-1 infection was assessed at day 3 after infection by intracellular p24 staining or GFP expression. For analysis of transmission of HIV-1 to T cells, LCs were stringently washed 3 days after infection followed by co-culture with activated allogeneic CD4+ T cells for 3 days. Triple staining with CD1a (LCs marker), CD3 (T cells marker; 552851-PercP, BD Pharmigen) and p24 was used to discriminate the percentage of CD3+CD1a−p24+ infected T cells. HIV-1 infection and transmission was assessed by FACSCanto II flow cytometer (BD Biosciences) and data analysis was carried out with FlowJo software (Treestar). HIV-1 production was determined by a p24 antigen ELISA in culture supernatants (ZeptoMetrix). mRNA was isolated with an mRNA Capture kit (Roche) and cDNA was synthesized with a reverse-transcriptase kit (Promega). For real-time PCR analysis, PCR amplification was performed in the presence of SYBR green in a 7500 Fast Realtime PCR System (ABI). Specific primers were designed with Primer Express 2.0 (Applied Biosystems; Extended Data Table 1). The cycling threshold (C ) value is defined as the number of PCR cycles in which the fluorescence signal exceeds the detection threshold value. For each sample, the normalized amount of target mRNA (N ) was calculated from the C values obtained for both target and household (GAPDH, primary LCs, DCs and U87 cells lines; β-actin, MUTZ-LCs) mRNA with the equation N  = 2Ct(control) − Ct(target). For relative mRNA expression, control siRNA sample was set at 1 within the experiment and for each donor. A two-step Alu-long terminal repeat (LTR) PCR was used to quantify the integrated HIV-1 DNA in infected cells as previously described20. Total cell DNA was isolated at 16 h after infection (multiplicity of infection of 0.4) with a QIAamp blood isolation kit (Qiagen). In the first round of PCR, the DNA sequence between HIV-1 LTR (LTR R region, extended with a marker region at the 5′ end) and the nearest Alu repeat was amplified (primer sequences, Extended Data Table 1). The second round was nested quantitative real-time PCR of the first-round PCR products using primers annealing to the aforementioned marker region in combination with another HIV-1-specific primer (LTR U5 region) by real-time quantitative PCR. Two different dilutions of the PCR products from the first-round of PCR were assayed to ensure that PCR inhibitors were absent. For monitoring the signal contributed by unintegrated HIV-1 DNA, the first-round PCR was also performed using the HIV-1-specific primer (LTR R region) only. HIV-1 integration was normalized relative to GAPDH DNA levels. For relative HIV-1 integration, control siRNA-infected cells (total signal; Supplementary Table 1) was set as 1 for one experiment or for each donor. A BlaM-Vpr-based assay was used to quantify fusion of HIV-1 to the host membrane in infected LCs as previously described10. LCs were infected with NL4.3-BlaM-Vpr for 2 h and then loaded with CCF2/AM (1 mM, LiveBLAzer FRET-B/G Loading Kit, Life technologies) in serum-free IMDM medium for 1 h at 25 °C. After washing, BlaM reaction was allowed to develop for 16 h at 22 °C in IMDM supplemented with 10% FCS and 2.5 mM anion transport inhibitor probenecid (Sigma Pharmaceuticals). HIV-1 fusion was determined by monitoring the changes in fluorescence of CCF2/AM dye, which reflect the presence of BlaM-Vpr into the cytoplasm of target cells upon viral fusion. The shift from green emission fluorescence (500 nm) to blue emission fluorescence (450 nm) of CCF2/AM dye was assessed by flow cytometer LSRFortessa (BD Biosciences) and data analysis was carried out with FlowJo software. Percentages of blue fluorescent CCF2/AM+ cells are depicted as percentage of HIV-1 fusion. A fluorescent bead adhesion assay was used to examine the ability of HIV-1 gp120-coated fluorescent beads to bind Langerin in CD4+CCR5+ U87 transfectants as previously described5. Binding was measured by FACSCanto II flow cytometer and data analysis was carried out with FlowJo software. Skin LCs and DCs were transfected with 50 nm siRNA with the transfection reagent DF4 (Dharmacon) whereas MUTZ-LCs, CD4+CCR5+ U87 parental or transduced cells were transfected with transfection reagent DF1 (Dharmacon) and were used for experiments 48–72 h after transfection. The siRNA (SMARTpool; Dharmacon) were specific for Atg5, (M-004374-04), Atg16L1 (M-021033), LSP-1 (M-012640-00), TRIM5α (M-007100-00) and non-targeting siRNA (D-001206-13) served as control. Langerin was silenced in MUTZ-LCs by electroporation with Neon Transfection System (ThermoFischer Scientific) using siRNA Langerin (10 μM siRNA, M-013059-01, SMARTpool; Dharmacon). Silencing of the aforementioned targets was verified by real-time PCR, flow cytometer and immunoblotting (Extended Data Figs 1d, e, 2a–k). Cells were pre-treated with bafilomycin A1 for 2 h or left untreated followed by incubation with HIV-1 for 16 h. Quantification of intracellular LC3 II levels by saponin extraction was performed as described before34, 35. LCs were washed in PBS and permeabilized with 0.05% saponin in PBS. Cells were incubated at 4 °C for 30 min with mouse anti-LC3 primary antibody (M152-3; MBL International) or with mouse anti-IgG1 isotype control (MOPC-21; BD Pharmingen) followed by incubation with Alexa Fluor 488-conjugated goat-anti mouse IgG antibody (A-21121, Life Technologies) in saponin buffer. Intracellular LC3 II levels were assessed by FACSScan or FACSCanto II flow cytometers (BD Biosciences) and data analysis was carried out with FlowJo. Cells were pre-treated with bafilomycin for 2 h or left untreated followed by incubation with HIV-1 for 4 h. Quantification of intracellular LC3 II levels by saponin extraction was performed as described before35. Whole-cell extracts were prepared using RIPA lysis buffer supplemented with protease inhibitors (9806; Cell Signalling). 20–30 μg of extract were resolved by SDS–PAGE (15%) and immunoblotted with LC3 (2G6; Nanotools) and β-actin (sc-81178; Santa Cruz) antibodies, followed by incubation with HRP-conjugated secondary rabbit-anti-mouse antibody (P0161; Dako) and luminol-based enhanced chemiluminescence (ECL) detection (34075; Thermo Scientific). For gel source data, see Supplementary Fig. 1. MUTZ-LCs (2 × 106) were incubated for 16 h with HIV-1 NL4.3 (multiplicity of infection, 0.5) or left untreated as a control, fixed in 4% paraformaldehyde and 1% glutaraldehyde in sodium cacodylate buffer for 10 min at room temperature followed by 24 h at 4 °C. After fixation, cells were collected by centrifugation and the pellet was washed in sodium cacodylate buffer. Cells were post-fixed for 1 h at 4 °C (1% osmium tetroxide, 0.8% potassium ferrocyanide in the same buffer), contrasted in 0.5% uranyl acetate, dehydrated in a graded ethanol series and embedded in epon LX112. Ultrathin sections were stained with uranylacetate/lead citrate and examined with a FEI Tecnai-12 transmission electron microscope. Numbers of autophagosomes per cell was determined in 50 cells for each condition counted by two independent researchers. LCs were left to adhere onto poly-l-lysine coated slides. Cells were fixed in 4% paraformaldehyde and permeabilized with PBS/0.1% saponin/1% BSA/1 mM Hepes. Cells were stained with anti-Langerin (AF2088; R&D Systems) and TRIM5α (ab109709; Abcam) antibodies followed by Alexa Fluor 647-conjugated anti-goat (A-21447; Life Technologies) and Alexa Fluor 488-conjugated anti-rabbit (A-21206; Life Technologies). For detection of autophagic vesicles, LCs were pre-loaded with the Cyto-ID Green detection autophagy reagent (ENZ-51031; Enzo Life Sciences), which was previously shown to specifically stain autophagic vesicles36 before adherence to microscope slides and stained with p24 (KC57-RD1-PE; Beckman Coulter) followed by Alexa-Fluor-546-conjugated anti-mouse (A-11003; Life Technologies). Nuclei were counterstained with Hoechst (10 μg ml−1; Molecular Probes). Single plane images were obtained by Leica TCS SP-8 X confocal microscope and data analysis was carried out with Leica LAS AF Lite (Leica Microsystems). Whole-cell extracts were prepared using RIPA lysis buffer supplemented with protease inhibitors. Atg16L1, DC-SIGN, Langerin, p62 and TRIM5α were immunoprecipitated from 40 μg of extract with anti- Atg16L1 (PM040; MBL International), DC-SIGN (AZN-D1)19, Langerin (10E2)5, p62 (ab56416; Abcam), TRIM5α (ab109709; Abcam), mouse IgG1 isotype control (MOPC-21; BD Pharmingen), mouse IgG2a isotype control (IC003A; R&D systems) and rabbit IgG control (sc-2077; Santa Cruz) coated on protein A/G PLUS agarose beads (sc-2003; Santa Cruz), washed twice with ice-cold RIPA lysis buffer and resuspended in Laemmli sample buffer (161-0747, Bio-Rad). Immunoprecipitated samples were resolved by SDS–PAGE (12.5%), and detected by immunoblotting with Atg5 (PM050; MBL), Atg16L1 (MBL), DC-SIGN (551186; BD Biosciences), Langerin (AF2088; R&D Systems), LSP-1 (3812S; Cell Signalling), TRIM5α (Abcam) and HIV-p24 (KC57-RD1-PE; Beckman Coulter) antibodies, followed by incubation with Clean-Blot IP Detection Kit-HRP (21232; Thermo Scientific) and ECL detection (34075; Thermo Scientific). Data acquisition was carried out with ImageQuant LAS 4000 (GE Healthcare). Immunoprecipitation with TRIM5α, Langerin, DC-SIGN, Atg16L1 and p62 pulls-down mostly the TRIM5α (approximately 56 kDa) form. Relative intensity of the bands was quantified using Image Studio Lite 5.2 software by normalizing β-actin and set at 1 in untreated cells. For gel source data, see Supplementary Fig. 1. Two-tailed Student’s t-test for paired observations (differences of stimulations within the same donor or cell-type) or unpaired observation (differences between U87 transfectants). Statistical analyses were performed using GraphPad 6.0 software and significance was set at P < 0.05 (*P < 0.05; **P < 0.01). The data that support the findings of this study are available from the corresponding author upon reasonable request.


News Article | February 15, 2017
Site: www.nature.com

C57BL/6N mice, ICR mice and Wistar rats were purchased from SLC Japan (Shizuoka, Japan). All animals were maintained under specific pathogen-free conditions. All animal experiments were approved by the Insititutional Animal Care and Use Committee, and performed in accordance with the guidelines of the University of Tokyo and the National Institute for Physiological Sciences. Diabetes was induced in 8-week-old C57BL/6N male mice by intravenous injection of 150 mg kg−1 of STZ. Mice with nonfasting blood glucose levels over 350 mg dl−1 1 week after STZ administration were used. Embryo culture and manipulation are described11. Rodent islets conventionally are isolated by collagenase perfusion of the pancreata through the common bile duct. However, the pancreata of Pdx1mu/mu chimaeric rats could not be perfused in this way because the pancreaticobiliary junction was maldeveloped in all (Extended Data Fig. 5). Therefore, we isolated islets by digestion of minced pancreata with collagenase. Pancreata removed from interspecific chimaera were inflated by interstitial injection of Gey’s balanced salt solution (GBSS; Sigma-Aldrich). GBSS-filled pancreata were minced using scissors. Small pieces of chopped pancreata were digested with collagenase XI (Sigma-Aldrich) to release islets from exocrine tissue. After 6–8 min incubation, islets were picked up using glass micropipettes and transplanted beneath the kidney capsule of 10-week-old male mice with STZ-induced diabetes, as previously described11. To prevent acute graft rejection, 0.5 mg per g (body weight) per day of tacrolimus, was injected intraperitoneally on the day of transplantation and on each of the following 4 days, in addition to an anti-inflammatory cocktail (all components, Affymetrix) containing anti-mouse interferon-γ mAb (rat IgGκ, 16-7312, clone R4-6A2), anti-mouse tumour necrosis factor-α mAb (rat IgG1κ, 16-7322, clone MP6-XT3) and anti-mouse IL-1β (hamster IgG, 16-7012, clone B122). The mRNA of TALENs (left and right) and rat Exo1 were generated by in vitro transcription. Linearized plasmids were transcribed from T7 promoter using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific) and resultant mRNAs were cleaned up by MEGAclear Kit (Thermo Fisher Scientific). 3 or 10 ng μl−1 of each mRNA was prepared by dilution in RNase- free-water and mixture of right TALEN, left TALEN and Exo1 were injected into the male pronuclei of zygotes by microinjection, as previously reported18. TALEN potential off-target sites were predicted by TALENoffer software. We chose 21 candidates (5 in exonic loci, 13 in intronic loci, 3 in intergenic loci) from TOP200 candidates19. We performed PCR amplification of genomic DNA from Pdx1+/muA, Pdx1+/muB and wild-type Wistar rats, subjecting the amplicons to Sanger sequencing. Genomic DNA was isolated from fluorescent-marker-negative cells isolated by FACS from chimaeric-rat blood samples. The TALEN target region of Pdx1 was amplified by PCR using the following primers: (forward) 5′-GCTGAGAGTCCGTGAGCTGCCCAG-3′ and (reverse) 5′-GGAACGCTTAAAGATCGTAGCAGC-3′). The PCR products were sequenced. Total RNA was isolated from duodenum of Pdx1muA/muB mice and reverse-transcribed by Superscript III reverse transcriptase (Thermo Fisher Scientific) with oligodT primer. Pdx1muA or Pdx1muB full-length cDNA were amplified by PCR using the following primers: (forward) 5′-GGCGCTGAGAGTCCGTGAGCTGC-3′ and (reverse) 5′-TTTTTTTTTTTTTTTGAAACCTCAAACAG-3′. Nonfasting blood glucose levels were determined (Medisafe-Mini glucometer; Terumo) weekly after islet transplantation. GTTs in overnight-fasted chimaera rats was conducted 0, 15, 30, 60 and 120 min after intraperitoneal injection of glucose (50% d-glucose solution, 2.5 g per kg body weight). Tail-vein blood was sampled by phlebotomy. Non-fasting serum mouse or rat c-peptide levels were analysed by enzyme-linked immunosorbent assay (ELISA) (mouse c-peptide ELISA kit, Shibayagi and Morinaga Institute of Biological Science; rat c-peptide ELISA kit, MERCODIA AB). Serum was isolated from 10-week-old Pdx1muA/muB + mPSCs chimaeras, C57BL/6N mice and Wistar rats. Serum was obtained from STZ-treated diabetic mice transplanted with mouseR islets 260 or 372 days after transplantation. SGE2 (EGFP-expressing mES cells) were derived from blastocysts generated from mating C57BL/6N female mice with C57BL/6N-Tg male mice (CAG-EGFP) (SLC Japan). mRHT (mES cells) were derived from blastocysts generated from mating male and female H2B-tdTomato knock-in mice with human histone H2B and tdTomato fusion gene in the mouse ROSA locus (T.K., unpublished data). Wlv3i-1 (rES cells) and GT3.2 (miPSCs) have been previously described11, 31. Maintenance of mPSCs and rPSCs has been previously described32, 33. Briefly, mPSCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 1,000 units per ml of mouse leukaemia inhibitory factor (all Thermo Fisher Scientific) and 1% l-glutamine-penicillin-streptomycin (Sigma-Aldrich). rPSCs were cultured on mitomycin-C-treated mouse embryonic fibroblasts in N2B27 medium supplemented with 1 μM mitogen-activated protein kinase inhibitor PD0325901 and 3 μM glycogen synthase kinase inhibitor CHIR99021 (both Axon Groeningen). All PSC lines were authenticated by chimaera formation. These cell lines were not contaminated with mycoplasma. Isolated pancreata and islets were fixed in 4% paraformaldehyde in phosphate-buffered saline solution (PBS). Paraffin-embedded sections were incubated with blocking buffer (Active Motif) for 1 h at room temperature. The sections were incubated with primary antibodies, diluted in blocking buffer for 1 h at room temperature, and washed three times with PBS. They were then incubated with secondary antibodies for 1 h at room temperature. Primary antibodies used were guinea pig anti-insulin (Abcam; ab7842), rabbit anti-glucagon (Nichirei Bioscience, 422271), rabbit anti-somatostatin (Nichirei Bioscience 422651), rabbit anti-cytokeratin 19 (Abcam; ab52625, clone EP1580Y), mouse anti-amylase (SantaCruz; SC-46657, clone G-10) and goat anti-GFP (Abcam; ab6673), with Alexa-488-, Alexa-546-, and Alexa-633-conjugated secondary antibodies (Thermo Fisher Scientific). After antibody treatment, sections were mounted with Vectashield (Vector Laboratories), a mounting medium containing DAPI (Thermo Fisher Scientific) for nuclear counterstaining, and sections were observed under fluorescence microscopy. Three to five sections per slide were imaged and processed using Image J. For detection of lymphoid infiltration, DAB immunohistochemistry was performed with rabbit anti-CD3 (Abcam; ab5690) and rabbit anti-CD11b (Bioss Inc.; bs-1014R). Islets or small pieces of kidney that included transplanted islets were dispersed into single cells with collagenase type1A (Sigma-Aldrich). Dispersed cells stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (Thermo Fisher Scientific; A16201, clone 390) or allophycocyanin (APC)-conjugated anti-rat CD31 (Thermo Fisher Scientific; 50-0310-82, clone TLD-3A12) were subjected to FACS CantoII analysing (BD Biosciences). Data were collected for all of the dispersed cells and analysed. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Sample size was estimated on the basis of previous publications. Statistical significance was calculated by F-test and Student’s t-test (compare two groups) and the similarity to the Mendelian ratio was analysed by chi-square test (with Excel and Graphpad Prism software). P < 0.05 was considered to be statistically significant. Data are presented as mean ± s.d. Immunohistochemistry and flow-cytometry studies were repeated three times independently with similar results. All relevant data that are included with this study are available from corresponding auther upon reasonable request.


News Article | February 22, 2017
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Pre-B acute lymphoblastic leukaemia (ALL) cells were obtained from patients who gave informed consent in compliance with the guidelines of the Internal Review Board of the University of California San Francisco (Supplementary Table 2). Leukaemia cells from bone marrow biopsy of patients with ALL were xenografted into sublethally irradiated NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice via tail vein injection. After passaging, leukaemia cells were collected. Cells were cultured on OP9 stroma cells in minimum essential medium-α (MEMα; Invitrogen), supplemented with 20% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin. Primary chronic myeloid leukaemia (CML) cases were obtained with informed consent from the University Hospital Jena in compliance with institutional internal review boards (including the IRB of the University of California San Francisco; Supplementary Table 3). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 20% BIT serum substitute (StemCell Technologies); 100 IU/ml penicillin and 100 μg/ml streptomycin; 25 μmol/l β-mercaptoethanol; 100 ng/ml SCF; 100 ng/ml G-CSF; 20 ng/ml FLT3; 20 ng/ml IL-3; and 20 ng/ml IL-6. Human cell lines (Supplementary Table 2) were obtained from DSMZ and were cultured in Roswell Park Memorial Institute medium (RPMI-1640; Invitrogen) supplemented with GlutaMAX containing 20% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cell cultures were kept at 37 °C in a humidified incubator in a 5% CO atmosphere. None of the cell lines used was found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. All cell lines were authenticated by STR profiles and tested negative for mycoplasma. BML275 (water-soluble) and imatinib were obtained from Santa Cruz Biotechnology and LC Laboratories, respectively. Stock solutions were prepared in DMSO or sterile water at 10 mmol/l and stored at −20 °C. Prednisolone and dexamethasone (water-soluble) were purchased from Sigma-Aldrich and were resuspended in ethanol or sterile water, respectively, at 10 mmol/l. Stock solutions were stored at −20 °C. Fresh solutions (pH-adjusted) of methyl pyruvate, OAA, 3-OMG (an agonist of TXNIP), d-allose (an agonist of TXNIP) and recombinant insulin (Sigma-Aldrich) were prepared for each experiment. DMS was obtained from Acros Organics, and fresh solutions (pH-adjusted) were prepared before each experiment. For competitive-growth assays, 5 mmol/l methyl pyruvate, 5 mmol/l dimethyl succinate (DMS) and 5 mmol/l OAA were used. The CNR2 agonist HU308 was obtained from Cayman Chemical. To avoid inflammation-related effects in mice, bone marrow cells were extracted from mice (Supplementary Table 4) younger than 6 weeks of age without signs of inflammation. All mouse experiments were conducted in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Bone marrow cells were obtained by flushing cavities of femur and tibia with PBS. After filtration through a 70-μm filter and depletion of erythrocytes using a lysis buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage or subjected to further experiments. Bone marrow cells were cultured in IMDM (Invitrogen) with GlutaMAX containing 20% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. To generate pre-B ALL (Ph+ ALL-like) cells, bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-7 (PeproTech) and retrovirally transformed by BCR–ABL1. BCR–ABL1-transformed pre-B ALL cells were propagated only for short periods of time and usually not for longer than 2 months to avoid acquisition of additional genetic lesions during long-term cell culture. To generate myeloid leukaemia (CML-like) cells, the myeloid-restricted protocol described previously30 was used. Bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-3, 25 ng/ml recombinant mouse IL-6, and 50 ng/ml recombinant mouse SCF (PeproTech) and retrovirally transformed by BCR–ABL1. Immunophenotypic characterization was performed by flow cytometry. For conditional deletion, a 4-OHT-inducible, Cre-mediated deletion system was used. For retroviral constructs used, see Supplementary Table 5. Transfection of retroviral constructs (Supplementary Table 5) was performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM medium (Invitrogen). Retroviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env). Lentiviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pCDNL-BH and VSV-G or EM140. 293FT cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with GlutaMAX containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 25 mmol/l HEPES, 1 mmol/l sodium pyruvate and 0.1 mmol/l non-essential amino acids. Regular medium was replaced after 16 h by growth medium containing 10 mmol /l sodium butyrate. After incubation for 8 h, the medium was changed back to regular growth medium. After 24 h, retroviral supernatant was collected, filtered through a 0.45-μm filter and loaded by centrifugation (2,000g, 90 min at 32 °C) onto 50 μg/ml RetroNectin- (Takara) coated non-tissue 6-well plates. Lentiviral supernatant produced with VSV-G was concentrated using Lenti-X Concentrator (Clontech), loaded onto RetroNectin-coated plates and incubated for 15 min at room temperature. Lentiviral supernatant produced with EM140 was collected, loaded onto RetroNectin-coated plates and incubated for 30 min at room temperature. Per well, 2–3 × 106 cells were transduced by centrifugation at 600g for 30 min and maintained for 48 h at 37 °C with 5% CO before transferring into culture flasks. For cells transduced with lentiviral supernatant produced with EM140, supernatant was removed the day after transduction and replaced with fresh culture medium. Cells transduced with oestrogen-receptor fusion proteins were induced with 4-OHT (1 μmol/l). Cells transduced with constructs carrying an antibiotic-resistance marker were selected with its respective antibiotic. For loss-of-function studies, dominant-negative variants of IKZF1 (DN-IKZF1, lacking the IKZF1 zinc fingers 1–4) and PAX5 (DN-PAX5; PAX5–ETV6 fusion) were cloned from patient samples. Expression of DN-IKZF1 was induced by doxycycline (1 μg/ml), while activation of DN-PAX5 was induced by 4-OHT (1 μg/ml) in patient-derived pre-B ALL cells carrying IKZF1 and PAX5 wild-type alleles, respectively. Inducible reconstitution of wild-type IKZF1 and PAX5 in haploinsufficient pre-B ALL cells carrying deletions of either IKZF1 (IKZF1∆) or PAX5 (PAX5∆) were also studied. Lentiviral constructs used are listed in Supplementary Table 5. A doxycycline-inducible TetOn vector system was used for inducible expression of PAX5 in mouse BCR–ABL1 pre-B ALL. The retroviral constructs used are listed in Supplementary Table 5. To study the effects of B-cell- versus myeloid-lineage identity in genetically identical mouse leukaemia cells, a doxycycline-inducible TetOn-CEBPα vector system31 was used to reprogram B cells. Mouse BCR–ABL1 pre-B ALL cells expressing doxycycline-inducible CEBPα or an empty vector were induced with doxycycline (1 μg/ml). Conversion from the B-cell lineage (CD19+Mac1−) to the myeloid lineage (CD19−Mac1+) was monitored by flow cytometry. For western blots, B-lineage cells (CD19+Mac1−) and CEBPα-reprogrammed cells (CD19−Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively, following doxycycline induction. For metabolic assays, sorted B-lineage cells and CEBPα-reprogrammed cells were cultured (with doxycycline) for 2 days following sorting, and were then seeded in fresh medium for measurement of glucose consumption (normalized to cell numbers) and total ATP levels (normalized to total protein). To study Lkb1 deletion in the context of CEBPα-mediated reprogramming, BCR–ABL1-transformed Lkb1fl/fl pre-B ALL cells expressing doxycycline-inducible CEBPα were transduced with 4-OHT(1 μg/ml) inducible Cre-GFP (Cre-ERT2-GFP). Without sorting for GFP+ cells, cells were induced with doxycycline and 4-OHT. Viability (expressed as relative change of GFP+ cells) was measured separately in B-lineage (gated on CD19+ Mac1−) and myeloid lineage (gated on CD19− Mac1+) populations. To study whether Lkb1 deletion causes CEBPα-dependent effects on metabolism and signalling, Lkb1fl/fl BCR–ABL1 B-lineage ALL cells expressing doxycycline-inducible CEBPα or an empty vector were transduced with 4-OHT-inducible Cre-GFP. After sorting for GFP+ populations, cells were induced with doxycycline. B-lineage cells (CD19+ Mac1−) and CEBPα-reprogrammed cells (CD19− Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively. Sorted cells were cultured with doxycycline and induced with 4-OHT. Protein lysates were collected on day 2 following 4-OHT induction. For metabolomics, sorted cells were re-seeded in fresh medium on day 2 following 4-OHT induction and collected for metabolite extraction. For CRISPR/Cas9-mediated deletion of target genes, all constructs including lentiviral vectors expressing gRNA and Cas9 nuclease were purchased from Transomic Technologies (Supplementary Table 5; see Supplementary Table 6 for gRNA sequences). In brief, patient-derived pre-B ALL cells transduced with GFP-tagged, 4-OHT-inducible PAX5 or an empty vector were transduced with pCLIP-hCMV-Cas9-Nuclease-Blast. Blasticidin-resistant cells were subsequently transduced with pCLIP-hCMV-gRNA-RFP. Non-targeting gRNA was used as control. Constructs including lentiviral vectors expressing gRNA and dCas9-VPR used for CRISPR/dCas9-mediated activation of gene expression are listed in Supplementary Table 5. Nuclease-null Cas9 (dCas9) fused with VP64-p65-Rta (VPR) was cloned from SP-dCas9-VPR (a gift from G. Church; Addgene plasmid #63798) and then subcloned into pCL6 vector with a blasticidin-resistant marker. gRNA sequences (Supplementary Table 6) targeting the transcriptional start site of each specific gene were obtained from public databases (http://sam.genome-engineering.org/ and http://www.genscript.com/gRNA-database.html)32. gBlocks Gene Fragments were used to generate single-guide RNAs (sgRNAs) and were purchased from Integrated DNA Technologies, Inc. Each gRNA was subcloned into pCL6 vector with a dsRed reporter. Patient-derived pre-B ALL cells transduced with either GFP-tagged inducible PAX5 or an empty vector were transduced with pCL6-hCMV-dCas9-VPR-Blast. Blasticidin-resistant cells were used for subsequent transduction with pCL6-hCMV-gRNA-dsRed, and dsRed+ cells were further analysed by flow cytometry. For each target gene, 2–3 sgRNA clones were pooled together to generate lentiviruses. Non-targeting gRNA was used as control. To elucidate the mechanistic contribution of PAX5 targets, the percentage of GFP+ cells carrying gRNA(s) for each target gene was monitored by flow cytometry upon inducible activation of GFP-tagged PAX5 or an empty vector in patient-derived pre-B ALL cells in competitive-growth assays. Cells were lysed in CelLytic buffer (Sigma-Aldrich) supplemented with a 1% protease inhibitor cocktail (Thermo Fisher Scientific). A total of 20 μg of protein mixture per sample was separated on NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–20% Mini-PROTEAN TGX precast gels, and transferred onto nitrocellulose membranes (Bio-Rad). The primary antibodies used are listed in Supplementary Table 7. For protein detection, the WesternBreeze Immunodetection System (Invitrogen) was used, and light emission was detected by either film exposure or the BioSpectrum Imaging system (UPV). Approximately 106 cells per sample were resuspended in PBS blocked using Fc blocker for 10 min on ice, followed by staining with the appropriate dilution of the antibodies or their respective isotype controls for 15 min on ice. Cells were washed and resuspended in PBS with propidium iodide (0.2 μg/ml) or DAPI (0.75 μg/ml) as a dead-cell marker. The antibodies used for flow cytometry are listed in Supplementary Table 7. For competitive-growth assays, the percentage of GFP+ cells was monitored by flow cytometry. For annexin V staining, annexin V binding buffer (BD Bioscience) was used instead of PBS and 7-aminoactinomycin D (7AAD; BD Bioscience) instead of propidium iodide. Phycoerythrin-labelled annexin V was purchased from BD Bioscience. For BrdU staining, the BrdU Flow Kit was purchased from BD Bioscience and used according to the manufacturer’s protocol. Methylcellulose colony-forming assays were performed with 10,000 BCR–ABL1 pre-B ALL cells. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers were counted after 14 days. Cell viability upon the genetic loss of function of target genes and/or inducible expression of PAX5 was monitored by flow cytometry using propidium iodide (0.2 μg/ml) as a dead-cell marker. To study the effects of an AMPK inhibitor (BML275), glucocorticoids (dexamethasone and prednisolone), CNR2 agonist (HU308), or TXNIP agonists (3-OMG and d-allose), 40,000 human or mouse leukaemia cells were seeded in a volume of 80 μl in complete growth medium on opaque-walled, white 96-well plates (BD Biosciences). Compounds were added at the indicated concentrations giving a total volume of 100 μl per well. After culturing for 3 days, cells were subjected to CellTiter-Glo Luminescent Cell Viability Assay (Promega). Relative viability was calculated using baseline values of cells treated with vehicle control as a reference. Combination index (CI) was calculated using the CalcuSyn software to determine interaction (synergistic, CI < 1; additive, CI = 1; or antagonistic, CI > 1) between the two agents. Constant ratio combination design was used. Concentrations of BML275, d-allose, 3-OMG and HU308 used are indicated in the figures. Concentrations of Dex used were tenfold lower than those of BML275. Concentrations of prednisolone used were twofold lower than those of BML275. To determine the number of viable cells, the trypan blue exclusion method was applied, using the Vi-CELL Cell Counter (Beckman Coulter). ChIP was performed as described previously33. Chromatin from fixed patient-derived Ph+ ALL cells (ICN1) was isolated and sonicated to 100–500-bp DNA fragments. Chromatin fragments were immunoprecipitated with either IgG (as a control) or anti-Pax5 antibody (see Supplementary Table 7). Following reversal of crosslinking by formaldehyde, specific DNA sequences were analysed by quantitative real-time PCR (see Supplementary Table 8 for primers). Primers were designed according to ChIP–seq tracks for PAX5 antibodies in B lymphocytes (ENCODE, Encyclopedia of DNA Elements, GM12878). ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown. CD19 and ACTA1 served as a positive and a negative control gene, respectively. The y axis represents the normalized number of reads per million reads for peak summit for each track. The ChIP–seq peaks were called by the MACS peak-caller by comparing read density in the ChIP experiment relative to the input chromatin control reads, and are shown as bars under each wiggle track. Gene models are shown in UCSC genome browser hg19. Extracellular glucose levels were measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen), according to the manufacturer’s protocol. Glucose concentrations were measured in fresh and spent medium. Total ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer’s protocol. In fresh medium, 1 × 106 cells per ml were seeded and treated as indicated in the figure legends. Relative levels of glucose consumed and total ATP are shown. All values were normalized to cell numbers (Figs 1b, c, 2c (glucose uptake), 3a and Extended Data Figs 2c, 4f, 6d) or total protein (Fig. 2c, ATP levels). Numbers of viable cells were determined by applying trypan blue dye exclusion, using the Vi-CELL Cell Counter (Beckman Coulter). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Flux Analyzer with an XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions. All compounds and materials were obtained from Seahorse Bioscience. In brief, 1.5 × 105 cells per well were plated using Cell-Tak (BD Biosciences). Following incubation in XF-Base medium supplemented with glucose and GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, OCR was measured at the resting stage (basal respiration in XF Base medium supplemented with GlutaMax and glucose) and in response to oligomycin (1 μmol/l; mitochondrial ATP production), mitochondrial uncoupler FCCP (5 μmol/l; maximal respiration), and respiratory chain inhibitor antimycin and rotenone (1 μmol/l). Spare respiratory capacity is the difference between maximal respiration and basal respiration. ECAR was measured under specific conditions to generate glycolytic profiles. Following incubation in glucose-free XF Base medium supplemented with GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, basal ECAR was measured. Following measurement of the glucose-deprived, basal ECAR, changes in ECAR upon the sequential addition of glucose (10 mmol/l; glycolysis), oligomycin (1 μmol/l; glycolytic capacity), and 2-deoxyglucose (0.1 mol/l) were measured. Glycolytic reserve was determined as the difference between oligomycin-stimulated glycolytic capacity and glucose-stimulated glycolysis. All values were normalized to cell numbers (Extended Data Fig. 2c) or total protein (Extended Data Figs 3a, 7a, b 8f) and are shown as the fold change relative to basal ECAR or OCR. Metabolite extraction and mass-spectrometry-based analysis were performed as described previously34. Metabolites were extracted from 2 × 105 cells per sample using the methanol/water/chloroform method. After incubation at 37 °C for the indicated time, cells were rinsed with 150 mM ammonium acetate (pH 7.3), and 400 μl cold 100% methanol (Optima* LC/MS, Fisher) and then 400 μl cold water (HPLC-Grade, Fisher) was added to cells. A total of 10 nmol norvaline (Sigma) was added as internal control, followed by 400 μl cold chloroform (HPLC-Grade, Fisher). Samples were vortexed three times over 15 min and spun down at top speed for 5 min at 4 °C. The top layer (aqueous phase) was transferred to a new Eppendorf tube, and samples were dried on Vacufuge Plus (Eppendorf) at 30 °C. Extracted metabolites were stored at −80 °C. For mass spectrometry-based analysis, the metabolites were resuspended in 70% acetonitrile and 5 μl used for analysis with a mass spectrometer. The mass spectrometer (Q Exactive, Thermo Scientific) was coupled to an UltiMate3000 RSLCnano HPLC. The chromatography was performed with 5 mM NH AcO (pH 9.9) and acetonitrile at a flow rate of 300 μl/min starting at 85% acetonitrile, going to 5% acetonitrile at 18 min, followed by an isocratic step to 27 min and re-equilibration to 34 min. The separation was achieved on a Luna 3u NH2 100A (150 × 2 mm) (Phenomenex). The Q Exactive was run in polarity switching mode (+3 kV/−2.25 kV). Metabolites were detected based on retention time (t ) and on accurate mass (± 3 p.p.m.). Metabolite quantification was performed as area-under-the-curve (AUC) with TraceFinder 3.1 (Thermo Scientific). Data analysis was performed in R (https://www.r-project.org/), and data were normalized to the number of cells. Relative amounts were log -transformed, median-centred and are shown as a heat map. To generate a model for pre-leukaemic B cell precursors expressing BCR–ABL1, BCR–ABL1 knock-in mice were crossed with Mb1-Cre deleter strain (Mb1-Cre; Bcr+/LSL-BCR/ABL) for excision of a stop-cassette in early pre-B cells. Bone marrow cells collected from Mb1-Cre; Bcr+/LSL-BCR/ABL mice cultured in the presence of IL-7 were primed with vehicle control or a combination of OAA (8 mmol/l), DMS (8 mmol/l) and insulin (210 pmol/l). Following a week of priming, cells were maintained and expanded in the presence of IL-7, supplemented with vehicle control or a combination of OAA (0.8 mmol/l) and DMS (0.8 mmol/l) for 4 weeks. Pre-B cells from Mb1-Cre; Bcr+/LSL-BCR/ABL mice expressed low levels of BCR–ABL1 tagged to GFP, and were analysed by flow cytometry for surface expression of GFP and CD19. The methylcellulose colony-forming assays were performed with 10,000 cells treated with vehicle control or metabolites. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm diameter dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers counted after 14 days. For in vivo transplantation experiments, cells were treated with vehicle control or metabolites (OAA/DMS) for 6 weeks. One million cells were intravenously injected into sublethally irradiated (250 cGy) 6–8-week-old female NSG mice (n = 7 per group). Mice were randomly allocated into each group, and the minimal number of mice in each group was calculated by using the ‘cpower’ function in R/Hmisc package. No blinding was used. Each mouse was killed when it became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move). The bone marrow and spleen were collected for flow cytometry analyses for leukaemia infiltration (CD19, B220). After 63 days, all remaining mice were killed and bone marrow and spleens from all mice were analysed by flow cytometry. Statistical analysis was performed using the Mantel–Cox log-rank test. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Following cytokine-independent proliferation, BCR–ABL1-transformed Lkb1fl/fl or AMPKa2fl/fl pre-B ALL cells were transduced with 4-OHT-inducible Cre or an empty vector control. For ex vivo deletion, deletion was induced 24 h before injection. For in vivo deletion, deletion was induced by 4-OHT (0.4 mg per mouse; intraperitoneal injection). Approximately 106 cells were injected into each sublethally irradiated (250 cGy) NOD/SCID mouse. Seven mice per group were injected via the tail vein. We randomly allocated 6–8-week-old female NOD/SCID or NSG mice into each group. The minimal number of mice in each group was calculated using the ‘cpower’ function in R/Hmisc package. No blinding was used. When a mouse became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move), it was killed and the bone marrow and/or spleen were collected for flow cytometry analyses for leukaemia infiltration. Statistical analysis was performed by Mantel–Cox log-rank test. In vivo expansion and leukaemia burden were monitored by luciferase bioimaging. Bioimaging of leukaemia progression in mice was performed at the indicated time points using an in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen). d-luciferin (Promega) dissolved in PBS was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the luminescence signal. General anaesthesia was induced with 5% isoflurane and continued during the procedure with 2% isoflurane introduced through a nose cone. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Data are shown as mean ± s.d. unless stated. Statistical significance was analysed by using Grahpad Prism software or R software (https://www.r-project.org/) by using two-tailed t-test, two-way ANOVA, or log-rank test as indicated in figure legends. Significance was considered at P < 0.05. For in vitro experiments, no statistical methods were used to predetermine the sample size. For in vivo transplantation experiments, the minimal number of mice in each group was calculated through use of the ‘cpower’ function in the R/Hmisc package. No animals were excluded. Overall survival and relapse-free survival data were obtained from GEO accession number GSE11877 (refs 35, 36) and TCGA. Kaplan–Meier survival analysis was used to estimate overall survival and relapse-free survival. Patients with high risk pre-B ALL (COG clinical trial, P9906, n = 207; Supplementary Table 10) were segregated into two groups on the basis of high or low mRNA levels with respect to the median mRNA values of the probe sets for the gene of interest. A log-rank test was used to compare survival differences between patient groups. R package ‘survival’ Version 2.35-8 was used for the survival analysis and Cox proportional hazards regression model in R package for the multivariate analysis (https://www.r-project.org/). The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated to ensure reproducibility of the observations. Gel scans are provided in Supplementary Fig. 1. Gene expression data were obtained from the GEO database accession numbers GSE32330 (ref. 12), GSE52870 (ref. 37), and GSE38463 (ref. 38). Patient-outcome data were derived from the National Cancer Institute TARGET Data Matrix of the Children’s Oncology Group (COG) Clinical Trial P9906 (GSE11877)35, 36 and from TCGA (the Cancer Genome Atlas). GEO accession details are provided in Supplementary Tables 9 and 10. ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown in UCSC genome browser hg19. All other data are available from the corresponding author upon reasonable request.


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The data on TP53 mutations (including allele frequency) and CNVs in pan-tumours and AML are derived from The Cancer Genome Atlas (TCGA) data in the cBioPortal for Cancer Genomics (http://www.cbioportal.org/; accessed on 29 October 2014). Only sequenced samples with allele frequency information provided were included in our analysis. Considering potential normal tissue contamination, samples with TP53 mutation allele frequency above 0.6 were considered as a homozygous mutation. The SNP data were visualized in IGV and statistics for AML outcome were analysed in Prism 6. Since cBioPortal only has a few non-Hodgkin lymphoma cases available, we used published data to extract TP53 mutation and deletion information18, 30, 31, 32, 33, 34. Clinical outcomes were annotated from follow-up data available within the Gene Expression Omnibus GSE34171 series. CNV analysis was performed using published AML and DLBCL tumour copy number data in Affymetrix SNP Array 6.0 .cel format (http://cancergenome.nih.gov/)18, 35, 36, 37 according to GISTIC2.0 (ref. 14). Specifically, the following GISTIC parameters and values were used following the latest TCGA Copy Number Portal analysis version (3 November 2014 stddata__2014_10_17; http://www.broadinstitute.org/tcga/gistic/browseGisticByTissue): core GISTIC version 2.0.22; reference genome build hg19; amplification threshold 0.1; deletion threshold 0.1; high-level amplification threshold 1.0; high-level deletion threshold 1.0; broad length cut-off 0.50; peak confidence level 0.95; cap 1.5; gene-GISTIC, true; arm-level peel-off, true; significance threshold 0.25; join segment size 8; X chromosome removed, false; maximum segments per sample 2,000; minimum samples per disease 40. To create a conditional 11B3 chromosome deletion, the MICER strategy was used15. Briefly, MICER clones MHPN91j22 (centromeric to Sco1) and MHPP248j19 (telomeric to Alox12) (Sanger Institute) were introduced into AB2.2 ES cells (129S5 strain, Sanger Institute) by sequential electroporation, followed by G418 (neomycin; 180 μg ml−1) and puromycin (1 μg ml−1) selection, respectively. Successful recombination events were confirmed by Southern blotting using the hybridized probes designated in Supplementary Table 2 as described38. The cis- and trans-localizations of two loxP sites in doubly targeted ES cells were further distinguished by PCR with df-F and df-R, or dp-F and dp-R (Supplementary Table 2), respectively, after Adeno-cre infection and HAT (Gibco) selection. Correct cis-ES clones in which two loxP sites were integrated into the same allele were used to generate chimaera mice by blastocyst injection. The F1 pups were genotyped with 11B3-F and 11B3-R primers (Supplementary Table 2) and those positive backcrossed to C57BL/6 mouse strains for more than 10 generations. All of the mouse experiments were approved by the Institutional Animal Care and Use Committee at the Memorial Sloan Kettering Cancer Center. Eμ-Myc, Vav1-cre, Ella-cre, Trp53LSL-R270H/+, Trp53LSL-R72H/+, Trp53+/−, Trp53fl/+ and Rag1−/− mice were ordered from Jackson Laboratories21, 39, 40, 41, 42, 43, 44 and the Arf+/− mouse strain is a gift from C. Sherr45. Eμ-Myc mice with different Trp53 alterations were monitored weekly with disease state being defined by palpable enlarged solid lymph nodes and/or paralysis. Tumour monitoring was done as blinded experiments. For lymphoma generated by transplantation, 1 million Eμ-Myc HPSCs from embryonic day (E)13.5 fetal liver or autoMACS-purified B220+ B progenitor cells isolated from 6–8-week mouse bone marrow were transduced with retroviruses, followed by tail-vein injection into sublethally irradiated (6 Gy, Cs137) C57BL/6 mice (Taconic; 6–8-week old, female, 5–10 mice per cohort)11, 46. All recipient mice were randomly divided into subgroups before transplantation and monitored as described earlier. The generation of AML proceeded as previously reported29. Briefly, retrovirally infected c-Kit+ haematopoietic stem and progenitor cells were transplanted into sublethally irradiated (6 Gy, Cs137) C57BL/6 mice, followed by routine monitoring of peripheral blood cell counts and Giemsa–Wright blood smear staining. For secondary transplantation experiments, 1 million leukaemia cells were transplanted into sublethally irradiated (4.5 Gy) mice. The immunophenotypes of resulting lymphomas and leukaemias were determined by flow cytometry as previously reported using antibodies purchased from eBioscience11, 29. Statistical analysis of all survival data was carried out using the log-rank test from Prism 6. No statistical methods were used to predetermine sample size. MSCV-Myc-IRES-GFP and MLS-based retroviral constructs harbouring a GFP or mCherry fluorescent reporter and targeting Ren, Trp53, Eif5a, Nf1 or Mll3 have all been reported before11, 29, 47. For the tandem shRNA experiments performed in Fig. 3, mirE-based shRNAs targeting two different genes were cloned into an MLS-based vector in an analogous fashion to what has been previously described48, 49. Retrovirus packaging and infection of HSPCs was done as previously reported11, 29. B220+ cells were isolated from the bone marrow of 6-week-old Eμ-Myc mice by autoMACS positive selection with anti-B220 microbeads (Militeny Biotech). After overnight culture, cells were infected with retroviruses carrying the indicated shRNAs. Two days after infection, 0.5 × 106 cells were washed with PBS followed by annexin V buffer (10 mM HEPES, 140 mM NaCl, 25 mM CaCl , pH 7.4), and incubated at room temperature with Pacific Blue annexin V (BD Biosciences) and propridium iodide (PI; 1 μg ml−1; Sigma-Aldrich) for 15 min and analysed on a LSR II flow cytometer (BD Biosciences). For arachidonic acid treatment, pre-B cells were cultured out from bone marrow cells in pre-B cell medium (RPMI1640, 10% FBS, 1% penicillin/streptomycin, 50 μM β-mercaptoethanol, 3 ng ml−1 IL-7). After 3 days culture, pre-B cells were treated with a series concentration of arachidonic acid (Cayman Chemical) for 20 h, followed by annexin V staining as described earlier. Lymphoma cells isolated from lymph nodes of diseased animals were treated with vehicle (PBS) or 1 μg ml−1 adriamycin for 4 h. Whole cell lysates were extracted in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (Roche), followed by SDS–PAGE gel electrophoresis and blotting onto PVDF membranes (Millipore). Eμ-Myc;Arf−/− lymphoma cell lines were used as a positive control for p53 induction. The p53 antibody used was obtained from Novocastra (NCL-p53-505) and horseradish peroxidase (HRP)-conjugated β-actin antibody from Sigma (AC-15). Alox15b expressions were examined in NIH3T3 cells, which were infected by shRNAs targeting Ren or Alox15b and then selected by G418. Anti-Alox15b antibody is from Sigma (SAB2100110), and HRP-conjugated GAPDH antibody is from ThermoFisher Scientific (MA5-15738-HRP). RNA-seq and data analysis were performed by the Integrated Genomic and Bioinformatics core at the Memorial Sloan Kettering Cancer Center. Briefly, total RNA from 11B3fl/Trp53fl;shNf1;shMll3;Vav1-cre or Trp53fl/fl;shNf1;shMll3;Vav1-cre leukaemia cells (four lines per cohort), isolated from the bone marrow of moribund mice, was isolated by Trizol extraction (Life Technologies). After ribogreen quantification (Life Technologies) and quality control on an Agilent BioAnalyzer, 500 ng of total RNA (RNA integrity number > 8) underwent polyA selection and Truseq library preparation according to instructions provided by Illumina (TruSeq RNA Sample Prep Kit v.2) with 6 cycles of PCR. Samples were barcoded and run on a Hiseq 2500 in a 50 bp/50 bp paired-end run, using the TruSeq SBS Kit v.3 (Illumina). An average of 45 million paired reads were generated per sample. At the most the ribosomal reads represented 0.1% and the percentage of mRNA bases was close to 65% on average. The output from the sequencers (FASTQ files) was mapped to the mouse genome (mm9) using the rnaStar (https://code.google.com/p/rna-star/) aligner, with the two-pass mapping methods. After mapping, the expression counts of each individual gene were computed using HTSeq (http://www-huber.embl.de/users/anders/HTSeq), followed by normalization and differential expression analysis among samples using the R/Bioconductor package DESeq (http://www-huber.embl.de/users/anders/DESeq). Gene set enrichment analysis (GSEA) was performed with Broad’s GSEA algorithm. A list of all primers used for PCR analysis is given in Supplementary Table 2. For detection and quantification of 11B3 recombination/deletion two methods were employed. In both cases genomic DNA (gDNA) was extracted from lymphoma or leukaemia cells using Puregene DNA purification kit (Qiagen). Initially, semi-quantitative PCR was used to detect the recombined 11B3 allele using primers df-F and df-R, generating a 2.2 kb product (Fig. 2d). The estimated frequency of recombination was determined by dropping gDNA from 11B3+/− into 11B3fl/+ at various ratios. For qPCR of the 11B3 deletion (Fig. 2e), SYBR Green PCR Master Mix (Applied Biosystems) was used and cycling and analysis was carried out on a ViiA 7 (Applied Biosystems). Primers 11B3-Q-F and 11B3-Q-R were used to detect the floxed allele, and to estimate the frequency of 11B3 deletion. Allelic frequency in UPD analysis (Extended Data Fig. 5a) was determined similarly, in this case with serial dilution of wild-type gDNA into DNase-free water to construct a standard curve. Two-tailed t-test is used for statistics analysis by Prism 6. For p21 gene expression examination by RT–qPCR, RNA was isolated with Trizol, cDNA was synthesized with SuperScript III First-Strand Synthesis System (Life Technologies) and qPCR was performed as described earlier with primers p21-Q-F and p21-Q-R. Trp53 exons (2–10) were amplified from genomic DNAs of 11B3-deleted lymphomas by PCR (see Supplementary Table 2 for primer sequences) and subjected to Sanger sequencing. Mutations were called only if detected in sequencing reads carried out in the forward and reverse direction. SNP analysis of isolated lymphoma (tumour) or tail (normal) genomic DNAs from the same tumour-bearing mouse were carried out by Charles River laboratory. Briefly, a SNP Taqman assay with competing FAM- or VIC-labelled probes was used to detect the relevant C57BL/6 and 129S SNPs (D11Mit4 and D11NDS16) as described previously50. Genomic DNA was extracted from freshly isolated lymphoma cells from one Eμ-Myc;11B3fl/+;Vav-cre mice. One microgram of DNA was sonicated (17 W, 75 s) on an E220 sonicator (Covaris). Samples were subsequently prepared using standard Illumina library preparation (end repair, poly A addition, and adaptor ligation). Libraries were purified using AMPure XP magnetic beads (Beckman Coulter), PCR enriched, and sequenced on an Illumina HiSeq instrument in a multiplexed format. Sequencing reads per sample were mapped using Bowtie with PCR duplicates removed. Approximately 2.5 million uniquely mappable reads were further processed for copy number determination using the ‘varbin’ algorithm51, 52 with 5,000 bins, allowing for a median resolution of ~600 kb. GC content normalization, segmentation and copy number estimation was calculated as described53. A custom shRNA library was designed to target mouse homologues (six shRNAs for one gene) to all human protein-coding genes on chromosome 17p13.1 from ALOX12 to SCO1, except TP53 and EIF5A. shRNAs were cloned into a retrovirus-based vector MLS by pool-specific PCR as previously described11. Eμ-Myc HSPCs infected with pooled shRNAs were transplanted into sublethally irradiated recipient mice. Resulting tumours were harvested, and used to extract contained shRNAs, followed by HiSeq in HiSeq 2500 (Illumina). Twenty-two oligonucleotides of shRNAs used in this study are listed in Supplementary Table 3. Total lipids were extracted using Folch’s method54 and analysed by LC-MS as previously described55. Briefly, freshly harvested cells were homogenized by chloroform/methanol (2:1, v-v). After being washed by water, the lipid-containing chloroform phase is evaporated. Dried lipids were dissolved in 100 μl 95% acetonitrile (in H O), sonicated for 3–5 min, and spiked with 10 μl of 500 ng ml−1 deuterated internal standard solution (IS; arachidonic acid-d8; Cayman Chemical, 390010). Then, 5 μl samples were injected into Acquity ultra performance liquid chromatography (UPLC) system (Waters), equipped with Acquity UPLC BEH C18 column (100 mm × 2.1 mm I.D., 1.7 μm; Waters). Samples were washed through the column with a gradient 0.1% formic acid: acetonitrile mobile elution from 35:65 (v:v) to 5:95 for 10 min. Flow rate was 0.25 ml min−1. Right after HPLC, samples were analysed in a Quattro Premier EX triple quadrupole mass spectrometer (Waters), which has electrospray negative mode and MasslynxV4.1 software. For each run, a standard curve was generated with different concentration of arachidonic acid lipid maps MS standard (Cayman Chemical, 10007268) mixed with IS (50 ng ml−1 final concentration). Arachidonic acid standard m/z is 303.2, and IS is 311.3. Three Eμ-Myc lymphoma cell lines generated from Trp53fl/+;Vav1-cre or 11B3fl/+;Vav1-cre tumour-bearing mice were cultured in BCM medium (45% DMEM, 45% IMDM, 10% FBS, 2 mM glutamine, 50 μM β- mercaptoethanol, 1× penicillin/streptomycin) in 96-well plates. Cells were treated with the indicated concentrations of 4-hydroxycyclophosphamide (Toronto Research Chemicals) or vincristine (Bedford Laboratories) for 3 days. The number of living cells was determined by PI staining and cell counting on a Guava EasyCyte (EMD Millipore). Leukaemia cell lines from Trp53∆/∆ or 11B3∆/Trp53∆;shNf1;shMll3 mice were treated with cytarabine (araC; Bedford Laboratories) or JQ1 (a gift from J. Bradner) in stem cell medium (BCM medium supplemented with 1 ng ml−1 IL-3, 4 ng ml−1 IL-6 and 10 ng ml−1 SCF) and cell viability after 3 days was determined similarly. All cytokines are from Invitrogen.


The following antibodies and reagents were from BD Biosciences or eBiosciences: monoclonal antibodies (mAb) to CD11b-pacific-blue (M1/70), CD11c-APC, F4/80-FITC, CD3-pacific-blue, CD4-FITC, CD40-PE, CD80-PE, CD86-PE, CD40L-PE, CD69-PE, C5aR1-PE, and their corresponding isotypes antibodies (rat IgG2b pacific blue, Armenian hamster IgG-APC, rat IgG2a-PE, rat IgG2b PE), Fc blocking antibodies, and Cytofix/Cytopermkit. Anti-phospho-LAT (Tyr191), and anti-LAT clone 11B.12 were from Upstate cell signaling solutions. Rabbit GCS-specific antibody was from Abbiotec LLC. Rabbit affinity-purified GC-specific antibody was from Glycobiotech GmbH. The C5aR antagonist A8(Δ71−73) (C5aRA) was generated as described14. ELISA kits for the detection of human and mouse C5a and cytokines (IFNγ, TNF, IL-1β, IL-6, IL-12p40, IL-12p70, IL-17A/F, IL-23 and CCL18) were from R&D System or eBiosciences. Proteome Profiler A was from R&D System, anti-Profiler A, Bio-Rad Molecular Imager Gel Doc. Liberase Cl was from Roche. DNase (DNase), Diethanolamine (DEA), p-Nitrophenylphosphate (PNPP), MgCl , goat anti-mouse IgG2a, DNase-I kit, and anti-β actin antibody were from Sigma. Alkaline phosphatase-conjugated antibodies to mouse (IgG1, IgG2a/c, IgG2b, and IgG3), human IgG isotypes (IgG1, IgG2, IgG3, and IgG4), and rabbit IgG were from Southern Biotech. Tween 20, Nunc plates, Aminolink Plus Coupling Resin, and BCA protein assay reagents were from Thermo Scientific, RIPA buffer containing sodium orthovanadate and protease inhibitors were from Roche Diagnostics. GM-CSF and M-CSF were from Peprotech. Conduritol B epoxide (CBE) was from Calbiochem. Anti-CD11c, anti-CD11b and anti-CD4 microbeads were from Miltenyi Biotec. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG and biotinylated protein ladder detection pack were from Cell Signaling Technology Inc. GC and C12-GC standards were from Matreya, LLC and Avanti Polar lipids, Inc. The 4–12% BisTris gel, sample loading, reducing, running buffer, standard protein molecular weight marker, iBlot 2 dry blotting system, iBind western system, and enzyme-linked chemiluminescence (ECL) chemiluminescent substrate reagent kit, RPMI, DMEM, BSA, FBS, penicillin, streptomycin, HEPES, sodium pyruvate, Trizol, Gel apparatus, Xcell SureLock, and TRIzol reagent were from Invitrogen, Life Technology. RNeasy plus mini kit was from Qiagen. The U937 (ATCC CRL-1593.2TM) cell line, dimethylsulfoxide, and growth medium were from American Type Culture Collection. The U937 cell line has been thoroughly tested and authenticated by the supplier through DNA profiling. It has not been tested for mycoplasma contamination. High capacity RNA-cDNA kit, Taqman universal mastermixII, human and mouse pre-developed primer/probe sets for UGCG/Ugcg and Hypoxanthin phosphoribosyltransferase 1 (HPRT1/hprt) and the real-time PCR system (7500 fast) were from Applied Biosystem, Life Technology and Thermo Fisher Scientific, Inc. (NYSE: TMO). OCT freezing medium was from Sakura Finetek and Vectashield was from Vector Laboratories. The Fortessa-I, -II, and LSRII flow cytometers were from BD Biosciences. FCS Express software version 4 was from DeNovo Software. The plate reader was from Molecular Devices. The D409V/null mice (Gba19V/−) and wild-type controls were both on the mixed FVB/C57BL 6J/129SvEvBrd (50:25:25) backgrounds. Male and female mice were used at 20–24 weeks of age7. To directly assess the role of C5aR1-mediated effects, Gba19V/− mice were backcrossed to C5aR1-deficient mice for at least 10 generations. Out of these backcrosses, we generated double mutant mice (Gba19V/−C5ar1−/−) and Gba19V/−, wild-type and C5ar1−/− background-matched littermates. To assess the role of C5aR1, C5aR2 and FcγRs in pharmacologically induced Gaucher disease, wild-type mice and those lacking C5aR1, C5aR2, and activating FcγRs (Fcer1g−/−) or the inhibitory FcγRIIB (Fcgr2b−/−) of both sexes were used at ~12 weeks of age. Mice were bred and maintained in the specific-pathogen free facility at the Cincinnati Children’s Research foundation. Mice of the appropriate genotype were randomly assigned to groups. No specific randomization was performed. The investigators were not blinded to allocation during experiments and outcome assessment. Animal care was provided in accordance with National Institute of Health guidelines and was approved by Cincinnati Children’s Hospital Medical Center IACUC. Frozen sera from human patients with untreated Gaucher disease (n = 10) and healthy volunteers (n = 15) were de-identified. Patients with Gaucher disease were diagnosed at Cincinnati Children’s Hospital Medical Center. They did not receive any specific-enzyme therapy or substrate reduction therapy for Gaucher disease and are designated as untreated. The study was approved by the ethics committee at Cincinnati Children’s Hospital Medical Center. Protocols for human studies were approved by the Institutional Review Board, and patients with Gaucher disease and controls gave written, informed consent for the use of their serum for the studies described here. To assess the effect of genetic or pharmacological targeting of C5aR1 on the inflammatory response in Gaucher disease, wild-type (n = 10) and C5ar1−/− mice (n = 10) were treated with CBE, which is an irreversible inhibitor of acid β-glucosidase21. More specifically, both mouse strains were injected i.p. with 100 mg CBE per kg body weight or vehicle (PBS) per day for up to 60 days, which was the termination point of these experiments. After 60 days of the indicated treatment with CBE, immune cells (macrophages, DCs, and T cells) were purified from lung of these mouse strains and used for measurement of GC, costimulatory molecules, and several of the proinflammatory cytokines. In additional experiments, wild-type (n = 15) or Gba19V/− mice (n = 15) were injected with 100 μl of the C5aRA A8(Δ71−73) (i.p. 0.5 mg per kg) or vehicle (100 μl, PBS) on five consecutive days. Five days after the final C5aRA treatment, liver, spleen and lung were separated and measured for GC accumulation. In addition, DCs and CD4+ T cells were purified from the lung of the indicated mouse strains, co-cultured and measured for costimulatory molecule expression and the production of proinflammatory cytokines. Liver, spleen and lung of vehicle- or CBE-treated wild-type or C5ar1−/− mice were homogenized in 1% sodium taurocholate/1% Triton X-100. The protein concentrations of cells from such tissue lysates were determined by BCA assay using BSA as standard. GCase activities were determined fluorometrically with 4MU-Glc in 0.25% Na taurocholate and 0.25% Triton X-100 as described7. Liver, spleen, lung and bone marrow were collected aseptically. Single-cell suspensions from liver and lung were obtained from minced pieces that were treated with Liberase Cl (0.5 mg ml−1) and DNase (0.5 mg ml−1) in RPMI (45 min, 37 °C). Single-cell suspensions from spleen were obtained by grinding and then filtration through a 70-μm cell strainer. Similar suspensions of liver and lung were obtained from minced pieces that were treated with Liberase Cl (0.5 mg ml−1) and DNase (0.5 mg ml−1) in RPMI (45 min, 37 °C). For bone marrow cells, femurs, tibias and humeri were flushed with sterile PBS, followed by red blood cell lysis (155 mM NH Cl, 10 mM NaHCO , 0.1 mM EDTA), passage through a strainer. Cells were then pelleted by centrifugation at 350g. Viable cells were counted using a Neubauer chamber and trypan blue exclusion. DCs, macrophages and CD4+ T lymphocytes were purified from single-cell suspensions of liver, spleen and lung using CD11c, CD11b and CD4 (L3T4) microbeads according to the manufacturer’s protocol. The purity of the cells was ~90–95%. Bone marrow cells were used to differentiate macrophage as described22. Briefly, fresh bone marrow cells were stimulated with M-CSF (10 ng ml−1) in complete DMEM (FBS 10% + 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 10 mM HEPES and 1 mM sodium pyruvate). Cells were seeded in six-well tissue culture plates and incubated at 37 °C in a 5% CO atmosphere. Five days after cell seeding, supernatants were discarded and the attached cells were washed with 10 ml of sterile PBS. 10 ml of ice-cold PBS were added to each plate and incubated at 4 °C for 10 min. The macrophages were detached by gently pipetting the PBS across the dish. The cells were centrifuged at 200g for 5 min and resuspended in 10 ml of complete DMEM. The cells were counted, seeded and cultured for 12 h before they were used for further experiments. DCs were differentiated from bone marrow cells as described22. Briefly, bone marrow was flushed from the long bones of the limbs and depleted of red cells with ammonium chloride. Such bone marrow cells were plated in six-well plates (106 cells per ml, 3 ml per well) in RPMI 1640 medium supplemented with FBS (10%) and 100 U ml–1 penicillin, 100 μg ml−1 streptomycin, 10 mM HEPES and 1 mM sodium pyruvate and 10 ng ml−1 recombinant murine GM-CSF at days 0, 2, 4 and 6. Floating cells were gently removed and fresh medium was added. At day 7, nonadherent cells and loosely adherent proliferating DC aggregates were collected, counted, seeded and cultured for 12 h before they were used. Tissue cells were identified by flow cytometry. First, they were suspended in PBS containing 1% BSA. After incubation (15 min, 4 °C) with FcγR-blocking antibody 2.4G2, cells were stained (45 min, 4 °C) with the following antibodies to identify antigen-presenting cells and T cells: CD4 for T cells; CD11b and F4/80 for macrophages; and CD11b and CD11c for DCs. Cells were also stained with the respective isotype antibodies as controls. Macrophages were first identified by their typical FSC/SSC pattern, and F4/80 and CD11b expression. DCs were identified as CD11c+CD11b+ cells. Further, CD40, CD80 CD86 and C5aR1 expression was determined in tissue DCs. T cells were first characterized by their FSC/SSC pattern and CD3 staining. CD3+ T cells were further stained for CD4, CD40L and CD69 expression. A total of 106 events were acquired for each cell type isolated from the different organs. Specific surface expression was assessed relative to the expression of the corresponding isotype control antibody. Lipids were extracted from tissues (5 mg; liver, spleen, and lung), purified macrophages, DCs, CD4+ T lymphocytes, U937 cells and GC-specific IgG2a by chloroform and methanol1, 22. GC and GS species in IgG2a isolates were quantified by ESI-LC–MS/MS using a Waters Quattro Micro API triple quadrupole mass spectrometer interfaced with Acquity UPLC system7. Calibration curves were built for the GC species (C16:0, C18:0, C24:1) using C12-GC as standard. Quantification of GCs with various fatty acid chain lengths were realized by using the curve of each GC species with closest number of chain length. The total GCs in the tissues and purified IgG2a were normalized to 1 mg of tissue and protein, and immune cells to 1 × 106 cells. C5a concentrations were determined in sera or culture supernatants from bone-marrow-derived macrophages and DCs (each of 106 cells per 200 μl of complete RPMI media) of wild-type and Gba19V/− (n = 15 per group) mice, CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per of group), as well as in sera obtained from patients with untreated Gaucher disease (n = 10) and healthy control humans (n = 15) by commercial ELISA kits according to the manufacturer’s instructions. C5aR1 expression in macrophages and DCs purified from liver, spleen and lung of wild-type or Gba19V/− mice was evaluated by flow cytometry using a C5aR1-specific antibody. For detection of cytokines and chemokines, blood from CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per group) was obtained by cardiac puncture. Sera were isolated after one-hour incubation at room temperature. Sera were diluted 1:10 with sterile PBS and used for detection of cytokines and chemokines with Proteome Profiler A Densitometry, which was performed with a Bio-Rad Molecular Imager Gel Doc system. To assess the effect of C5a on GC-induced costimulatory molecule expression, DCs and CD4+ T cells purified from lungs of Gba19V/− mice and background-matched wild-type mice (n = 15 per group) were stimulated ex vivo in the presence or absence of different C5a concentrations (0, 8, 16 and 32 nM) for 24 h at 37 °C. DCs and CD4+ T cells were purified from liver, spleen and lung of CBE-treated C5ar1−/− and background-matched wild-type mice (n = 10 per group) and stained with CD40-, CD80- and CD86- (DCs) or CD40L- and CD69-specific antibodies (CD4+ T cells). To assess the effect of C5a on GC-induced cytokine and chemokine production, DCs and CD4+ T cells (1:2.5 ratio), purified from lungs of Gba19V/− mice and background-matched wild-type mice (n = 15 per group), were cocultured in the presence and absence of C5a (32 nM) for 48 h in complete medium. In additional experiments, indicated ratios (1:25) of DCs and CD4+ T cells, purified from lungs of CBE-treated and untreated wild-type and C5ar1−/− mice (n = 10 per each group), were cocultured for 48 h in complete medium. Supernatant of these experiments were used to determine IFNγ, TNF, IL-1β, IL-6, IL-12p40, IL-12p70, IL-17A/F and IL-23 by ELISA. To determine the levels of GC-specific IgG antibodies in mice and patients with Gaucher disease, 10 μg of GC were dissolved in 1 ml of methanol and water to a final concentration of 10 μg ml−1. 100 ml of this GC solution (1 μg per well) were used to coat a 96-well ELISA plate. GC-coated plates were kept overnight at room temperature followed by three washings with PBS containing 1% Tween-20 (PBST). Test sera (100 μl; 1:100) isolated from wild-type and Gba19V/− mice (n = 15 per group), CBE-treated and untreated wild-type mice (n = 10 per group), as well as healthy humans (n = 15) and untreated patients with Gaucher (n = 10), and GC-specific IgG control antibody were loaded into the lipid-coated wells, followed by incubation for 1.5 h at room temperature. These plates were then washed three times with PBST and subsequently incubated with alkaline phosphatase-conjugated rat anti-mouse IgG1 (1:500 in PBS), IgG2a/c (1:1,000 in PBS), IgG2b (1:1,000 in PBS) or IgG3 (1:1,000 in PBS) or alkaline phosphatase-conjugated mouse anti-human IgG1, IgG2 (each 1:1,000 in PBS), IgG3 and IgG4 (each 1:500 in PBS) in triplicates. Then, the plates were incubated for 1.5 h at room temperature followed by two washing steps with PBST and one with 10 mM DEA. 100 ml of 1 mg ml−1 PNPP in 10 mM DEA containing 5 mM MgCl was added to each well and incubated for 30 min at room temperature in the dark. Finally, plates were read at 405 nm to detect the GC-specific IgG antibodies. To determine GS- and GC-specific IgG IC formation, IgG2a was purified from pooled sera that were prepared from wild-type and Gba19V/− mice (n = 15 per group). Briefly, pooled mouse sera (5–10 ml) were incubated with goat anti-mouse IgG2a (25–50 μg) that had been immobilized on 2 ml of Aminolink Plus coupling resin overnight at 4 °C according to the manufacturer’s instructions. After several washing steps with working buffer (20 mM PBS, pH 7.4), bound IgG2a antibody fractions were finally eluted using 3 ml of elution buffer (50 mM Gly–HCl, pH 2.8). The eluted fractions were then used to determine GS and GC species bound to IgG2a and to quantify them with an ESI-LC–MS/MS system as above. Protein separation of purified IgG2a was performed using a 12% NuPAGE Bis-Tris Mini gel and reducing SDS–PAGE system according to the manufacturer’s instruction. Briefly, 4 μl of IgG2a (2.5 mg ml−1) was mixed with 16 μl of reducing buffer, (for example, 5 μl of NuPAGE LDS Sample Buffer 4×, 2 μl of NuPAGE Reducing Agent 10×, and 13 μl of deionized water) and then boiled for 5 min in a water bath. 10 μg of protein were applied to each lane and PAGE (130–180 mA) was run for 1 h at room temperature. The gel was then stained with Coomassie blue R250 using standard techniques. A minimum of two sections from CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per group), as well as C5aRA-treated and vehicle-treated wild-type and Gba19V/− mouse strains (n = 15 per group) were examined from each tissue. Liver, spleen and bone were collected after the mice had been perfused with PBS and the tissues fixed in 10% formalin or 4% paraformaldehyde, and processed for paraffin and frozen blocks, respectively. Paraffin sections of indicated tissues were stained with haematoxylin and eosin (H&E), whereas frozen sections were stained with rat anti-mouse CD68 (1:100) followed by biotinylated goat anti-rat and streptavidin-conjugated antibodies as described previously7, 23. To determine whether GC induces complement activation in Gaucher disease, we used freshly isolated liver, spleen and lung from CBE-treated and untreated wild-type and C5ar1−/− mice (n = 10 per group). These tissues were embedded in OCT freezing medium and snap-frozen in liquid nitrogen and eventually stored at −80 °C until use. Tissues were then sectioned at 5–7 μm and fixed with cold acetone and permeablized with 0.2% Triton X-100 in PBS. Tissue sections were blocked with 2% BSA and counter-stained with FITC-conjugated antibody to mouse C3/C3b (2 μg ml−1) and its isotype control overnight at 4 °C. Tissues were washed and coverslipped with Vectashield. Immunofluorescence images were captured with a Zeiss Apotome microscope (AxioV200). To investigate the direct effect of GC immune complexes on C5a release in Gaucher disease, macrophages (106 cells per 200 μl of complete RPMI media) purified from lung tissues of Gba19V/− mice (n = 15) were ex vivo stimulated in the presence or absence of GC (0.25, 0.5 and 1.0 μg) and anti-GC IgG (25 μg) for 2 h. Supernatants were used to determine C5a concentrations by ELISA. To evaluate the effect of GC immune complexes on C5a secretion in vivo, wild-type and Gba19V/− mice were injected i.p. with vehicle (ethanol), GC, anti-GC IgG or GC immune complexes (n = 15 per group). After 2 h, serum and peritoneal lavage fluid were collected and C5a was measured by ELISA according to the manufacturer’s instructions. After incubation of lung-derived F4/80+CD11b+ macrophages (5 × 106) from wild-type and Gba19V/− mice (n = 15 per group) with GC (1.0 μg), anti-GC IgG (25 μg of anti-GC IgG), GC immune complex or vehicle (1 μl methanol) per ml of media for 5 min at 37 °C, cells were collected and pellets were lysed with 1× RIPA buffer containing sodium orthovanadate and protease inhibitors. Protein concentrations were determined in cell lysates using BCA protein assay. Each 10 μg of cell lysates were loaded on an 10% SDS–PAGE and transferred onto a PVDF membrane and probed with antibodies to phosphorylated LAT (pLAT; 1:200) and non-phosphorylated LAT (linker of T cell activation; 1:1,000) using the iBlot 2 Gel transfer device and iBind western system according to the manufacturer’s instruction. pLAT and LAT (both ~36/38 kDa) proteins were visualized using anti-rabbit and anti-mouse secondary antibodies conjugated to HRP (1:1,000) and the Novex ECL chemiluminescent substrate reagent kit. Total RNA was extracted from mouse lung, liver, spleen and U937 macrophage-like cells using TRIzol reagent according to the manufacturer’s instructions. Reverse transcription was performed using the High capacity RNA to cDNAKit and qPCR was performed using Taqman assay reagents, primer/probes sets for both human and mouse UGCG/Ugcg and HPRT1/hprt. Amplifications were done using the ABI 7500 Real-Time PCR System and the calculations and analysis were based on the comparative C method24. For protein expression of GCS, mouse lung homogenates were lysed using mammalian protein extraction reagent. Protein concentration was determined by BCA according to the manufacturer’s instructions. Equal amounts of proteins (20 μg per lane) were loaded onto NuPAGE 4–12% Bis-Tris gradient SDS–PAGE. The mouse proteins were transferred to Hybond-ECL PVDF membranes and immunoblotted using iBind western blotting system with antibodies to mouse GCS diluted 1:100 with iBind solution and incubated over night at 4 °C. The GCS signals were detected using a HRP-conjugated anti-rabbit IgG (1:1,000) and ECL detection reagent as described25 with β-actin as a loading control. The intensities of protein bands were quantified using an NIH Image J. The GCS expression in the different treatment groups is depicted as the GCS/β-actin ratio normalized against the 100% value assigned to the wild-type group. To assess whether GC immune complexes causes C5a generation in patients with Gaucher disease, sera prepared from healthy humans (n = 15) and untreated patients with Gaucher disease (n = 10) were diluted 1:10,000 with saline and used to identify C5a by ELISA according to the manufacturer’s instructions. To determine the direct effect of GC immune complexes on C5a production and proinflammatory cytokine release in human Gaucher disease, the human macrophage-like cell line U937 (106 cells per 200 μl of complete RPMI media) was treated with CBE at 37 °C and 5% CO for 72 h. These cells were then stimulated in the presence or absence of GC (1 μg), anti-GC IgG (25 μg) or GC immune complex. Supernatants were used to determine C5a, CCL18, TNF, IL-1β, IL-6 and IL-23 concentrations by ELISA. All quantitative experiments were repeated at least three times. The sample sizes in all animal studies were estimated on the basis of effect sizes present in pilot studies to ensure we had sufficient power. The number of animals used in each experiment is outlined in the relevant sections in the Methods. An unpaired Student’s t-test (for two groups) or one-way analysis of variance (ANOVA) (for more than two groups) were used to determine significant differences between groups (Graph Pad Prism). To ensure that the statistical inference was appropriate, we evaluated the normality of the data distribution. Between group differences in many of the variables made, the overall distributions of many of the measures were non-normal. However, evaluation with non-parametric tests supported the inference of the parametric tests suggesting that the parametric tests were robust to these deviations from normality. For t- tests of the 40 cytokines, a simple Bonferroni correction is not appropriate as there is a high degree of correlation between the cytokines (average pairwise rho = 0.76). Thus, we employed a correlation corrected Bonferroni adjustment in SISA (http://www.quantitativeskills.com/sisa/calculations/bonfer.htm) resulting in a significance threshold of 0.021. As two conditions were considered for these 40 cytokines, the final correction was 0.021 / 2 = 0.0105. For the ANOVA, rather than considering all possible pairs of comparisons, we focused on a restricted set of a priori comparisons. Specifically, we performed analysis to determine the effect of (1) genotype, (2) C5aRA treatment, and (3) GCase targeting. Within each of these specific tests, we applied Bonferroni correction on the basis of the number of a priori comparisons made. For analyses, which were not pre-specified, the Bonferroni comparison was made on the number of possible comparisons. All data in the bar graphs are reported as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 denote the uncorrected P values, with the significance thresholds denoted in the figure legend if multiple testing corrections were applied. The data generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


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No statistical methods were used to predetermine sample size. Idelalisib (CAL-101, GS-1101; PI3Kδ inhibitor), duvelisib (IPI-145, INK1197; PI3Kγδ dual inhibitor), AS-604850 (PI3Kγ inhibitor) and ibrutinib (inhibitor of Bruton’s tyrosine kinase) were purchased from Selleckchem and all used at 1 μM concentration in most experiments. In some experiments, inhibitors were used at 0.1 μM or 0.5 μM concentrations, as indicated in the corresponding figure legend. Wild-type mice, c-myc25×I-SceI and c-myc25×I-SceIAicda−/− in the 129S2 mice background. All mice carrying the 25×I-SceI cassette were heterozygous for the modified c-myc allele containing the I-SceI cassette and were previously described9, 31. At least three independent mice of the same sex (females) and similar age (8–12 weeks) were used for each experiment with B cells. No mice were excluded from the analysis and no randomization or blinding method was used. Animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Boston Children’s Hospital (protocol 16-01-3093R) or by the Italian Ministry of Health for the University of Torino (approval no. 143/2013-B). They were housed and maintained in the specific-pathogen-free facility at Boston Children’s Hospital. Human leukaemia/lymphoma cell lines MEC1 (Chronic Lymphocytic Leukaemia), JeKo-1 and Mino (Mantle Cell Lymphoma), and GM06990 (EBV-immortalized lymphoblastoid B-cell line) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin (100 units per ml) and l-glutamine (2 mM). All cell lines tested negative for mycoplasma contamination. Cell lines were authenticated as they were purchased from ATCC (JeKo-1, Mino), DSMZ (MEC1) or the Coriell Institute (GM06990). For experiments with PI3K inhibitors, cells were plated in 6-well plates at a concentration of 5 × 105 cells per ml. Cells were collected at the indicated time points for RNA or protein isolation or flow cytometry analysis or after 4 days of treatment to isolate genomic DNA for HTGTS libraries. DNA before and after therapy from patients with CLL (untreated n = 8; idelalisib n = 10; ibrutinib n = 10; total 56 samples) was extracted from peripheral blood samples. Samples from idelalisib-treated patients were collected in the 99–224 CLL repository approved by the Dana-Farber Cancer Institute Institutional Review Board. Ibrutinib-treated patients were enrolled on a phase 2, open-label, single-centre, investigator-initiated study approved by the National Heart, Lung, and Blood Institutional Review Board at the National Institutes of Health (registered at http://www.clinicaltrials.gov, NCT01500733). All patients provided written informed consent. All cases were diagnosed according to the International guidelines and consented according to internal protocols. Details of treatment and sample collection for each patient are summarized in Supplementary Table 6. Splenic mouse B cells were isolated from mice by immunomagnetic depletion with anti-CD43 MicroBeads (Invitrogen) as previously described9. Briefly, all the non-B cells were depleted with anti-CD43 MicroBeads combined with Dynabeads Biotin Binder (Invitrogen); naive B cells were cultured at a concentration of 5 × 105 cells per ml in RPMI medium supplemented with 15% FBS, penicillin-streptomycin (100 units per ml), l-glutamine (2 mM), anti-CD40 (1 μg ml−1, eBioscience) and recombinant mouse IL-4 (20 ng ml−1; PeproTech). The purity of B-cell population was typically 96–98% in all experiments, as documented by flow cytometric analysis of B220 expression in enriched cells. Cells were collected after 4 days of treatment with inhibitors to isolate genomic DNA for HTGTS libraries and targeted re-sequencing experiments. For RNA and protein extraction, cells were collected at the indicated time points. Class switch recombination (CSR) was measured by staining with PE-labelled anti-mouse IgG (BD Biosciences) and Cy5-PE-labelled anti-mouse B220 (eBiosciences). Data acquisition was performed using a FACSVerse flow cytometer (BD biosciences). For immunization, sheep blood in Alsever’s solution (BD) were washed with PBS and re-suspended in PBS at a concentration of 1 × 109 sheep red blood cells per ml. 8–12-week-old mice were immunized by intraperitoneal injection of 2 × 108 sheep red blood cells in a 200 ml volume. After 5 days, a booster injection was performed using fivefold more sheep red blood cells. On day 6 and for 7 consecutive days, animals were daily administered vehicle (0.5% carboxymethylcellulose, 0.05% Tween 80 in ultra-pure water) or idelalisib or duvelisib (10 mg per kg per day) by oral gavage. Spleens were collected at the end of treatment, placed on ice, washed in PBS to remove residual blood, cut into small pieces, crushed and physically dissociated using a Falcon cell strainer, and subjected to hypotonic lysis of erythrocytes. Mouse germinal centre B cells were isolated from the spleens of immunized mice by immunomagnetic depletion: first non-B-cells were depleted with anti-CD43 MicroBeads; next enriched B cells were incubated with a cocktail of biotinylated antibodies specific for CD11c (eBiosciences) and IgD (eBiosciences) to remove dendritic cells and mature naive B cells, respectively, as previously reported32. Enrichment of the germinal B cells was evaluated with PE-labelled anti-mouse GL7 (eBiosciences) and Cy5-PE-labelled anti-mouse B220 (eBiosciences). 8-week-old female BALB/cAnNCrl mice were purchased from Charles River and housed in the University of Torino mouse facility under a protocol approved by the Italian Ministry of Health. Commercial pristane (2,6,10,14-tetramethylpentadecane) was purchased from Sigma. Pristane was administered by two 0.5 ml i.p. injections given 70 days apart, as previously described22. The mice were divided into four different groups: vehicle group (0.5% carboxymethylcellulose, 0.05% Tween 80 in ultra-pure water) and idelalisib or duvelisib or ibrutinib groups. Drugs were administered by oral gavage (10 mg per kg per day) for 70 days (5 days a week). Mice underwent follow-up assessment for the development of ascites and were killed when they reached a point of distress. Several tissues, including peritoneal tumour nodules, inflammatory granuloma, liver, spleen, intestine, were processed for histologic analysis. For histology, tissues and tumour nodules were fixed in 10% formalin over-night and transferred to 70% ethanol and embedded in paraffin. 4-μm-thick sections were stained with haematoxylin and eosin to evaluate the distribution of clusters of atypical plasma cells. Plasma cell tumours were diagnosed by finding clusters of 10 or more hyperchromatic, atypical plasma cells in hystology specimens, as previously reported22. PCR for Igh–c-myc translocations was performed on 500 ng of genomic DNA extracted from ascites by adapting protocols previously described33, 34. Briefly, we performed two rounds of PCR with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using primers listed in Supplementary Table 7. All PCR reactions were performed with appropriate positive controls (genomic DNA obtained from mouse B cells activated in vitro and treated with PI3Kδ inhibitors) and negative controls (DNA from Aicda−/− mouse B cells). PCR conditions were 98 °C for 30 s followed by 25 cycles (98 °C, 10 s; 62 °C, 30 s; 72 °C, 4 min) for both the first and second round. PCR amplicons were purified and sequenced to confirm Igh–c-myc translocations. Whole-cell extracts were obtained from purified mouse B cells or cell lines treated with 1 μM PI3K inhibitors using GST-FISH buffer (10 mM MgCl , 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES (pH 7.5)) supplemented with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na VO . Extracts were cleared by centrifugation at 12,000 r.p.m. for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 12% Mini-PROTEIN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), blocked with 5% skimmed milk (BIO-RAD). Primary antibodies for immunoblotting included: rat monoclonal anti-mouse-AID (mAID-2 clone, eBioScience, catalogue no. 14-5959-82), mouse monoclonal anti-human-AID (ZA001, Life Technologies, catalogue no. 39-2500), rabbit monoclonal anti-PI3K π110δ (Ψ387, Abcam, catalogue no. 32401), rabbit polyclonal anti-β−actin (Sigma, catalogue no. A2066), rabbit monoclonal anti-phospho-AKT (S473) (D9E, Cell Signaling Technology, catalogue no. 4060), rabbit monoclonal anti-AKT (pan) (C67E7, Cell Signaling Technology, catalogue no. 4691). Membranes were developed with ECL solution (GE Healthcare). AID protein abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane. Total RNA was isolated from primary mouse B cells and human lymphoma cells by TRIzol (Life Technologies). Before cDNA synthesis, 1 μg of total RNA was treated with 5 U μl−1 RNase-free recombinant DNase I (Roche). cDNA was transcribed using iScript cDNA synthesis kit following the manufacturer’s instructions (Biorad). All quantitative RT–PCR experiments were performed in triplicate on ICycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories) with SYBR green dye. Primer pairs are listed in Supplementary Table 7. Expression levels for individual transcripts were normalized against β-actin for murine samples or HuPO for human samples. Fold change in transcript levels were calculated as fold change over untreated cells. Retroviral supernatants were prepared from Phoenix packaging cells transfected with retroviral vectors. The pMX-I-SceI vector has been previously described9, PI3Kδ retroviruses (wild-type PI3K p110δ (denoted as p110δWT) and PI3K p110δ(E1021K)) were provided by K. Okkenhaug and F. Garcon (The Babraham Institute, UK)13. Briefly, Phoenix-ECO cells, a second-generation retrovirus-producer cell line, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin-streptomycin (100 units per ml) and l-glutamine (2 mM). To generate retroviral particle, 3.5 × 106 Phoenix-ECO cells were plated per 10-cm dish. The following day, cells were transfected by calcium phosphate transfection method with 10 μg of each plasmid and 5 μg of pCL-Eco retrovirus packaging plasmid. The media was changed 8 h after transfection. The viral supernatant was collected 48 h after transfection, passed through a 0.45 μm filter, pooled and used either fresh or snap-frozen. For transduction, one volume of viral supernatant with polybrene (6 μg ml−1) was added to mouse B cells after 24 h of activation with anti-CD40 plus IL-4, as previously described9. Plates were spun for 1.5 h at 2,400 r.p.m. and incubated overnight. Cells were washed and plated at a concentration of 5 × 105 cells per ml. On day 4 of stimulation, transduction efficiency was evaluated by measuring the percentage of transduced cells by enhanced green fluorescence protein expression (typical range was 50% to 85% of transduced cells). PI3K inhibitors were added at time of transduction and then maintained for the whole duration of the activation. CSR was evaluated by staining with Cy5-PE-labelled anti-mouse B220 (eBiosciences) and PE-labelled anti-mouse IgG (BD Biosciences). Data acquisition was performed using a FACSVerse flow cytometer (BD biosciences). CSR ranged between 15% and 40% for retrovirus-transduced B cells. DNA was isolated from cells at day 4 of culture according to standard methods for HTGTS libraries. For SpCas9 expression and generation of single guide RNA (sgRNA), the 20-nt target sequences were selected to precede a 5′-NGG protospacer-adjacent motif (PAM) sequence. The two c-MYC-targeting sgRNAs (1 and 2) and the AICDA sgRNA were designed with the CRISPR design tool from F. Zhang laboratory (http://crispr.mit.edu/). Oligonucleotides were purchased from Integrated DNA technology (IDT), annealed and cloned into the BsmbI-BsmBI sites downstream from the human U6 promoter in LentiCRISPR v2 plasmid (Addgene, 52961). Oligonucleotides used in this study for cloning are listed in Supplementary Table 7. HEK293FT cells (Invitrogen) were maintained in 10% FBS-containing DMEM. To generate lentiviral particles, 5.5 × 106 HEK293FT cells were plated per 10 cm dish. The following day, cells were transfected by calcium phosphate transfection method with 7.2 μg of lentiCRISPR v2 plasmid, 3.6 μg of VSVG, 3.6 μg of RSV-REV, and 3.6 μg of PMDLg/pPRE. The media was changed 8 h after transfection. The viral supernatant was collected 36 h after transfection, passed through a 0.45 μm filter, pooled and used either fresh or snap-frozen. For transduction of JeKo-1 and MEC1 with c-MYC CRISPR/Cas9 lentiviruses, a total number of 4 × 105 human neoplastic cells were plated into 6-well plates, at a concentration of 2 × 105 cells per ml. Lentiviral transduction was performed adding lentiviral supernatant, spinning for 1.5 h at 2,400 r.p.m. in the presence of polybrene (6 μg ml−1). The viral supernatant was exchanged for fresh medium 8 h later. PI3K inhibitors were added 8 h before the infection and after washing. After 48 h, cells were selected with 0.2 μg ml−1 of puromycin to enrich for transduced cells. The cells were collected after 3 days from the puromycin addition. Genomic DNA was extracted as previously described for HTGTS libraries. To generate the AID-knockout MEC-1 cell line, MEC-1 cells were transduced with AID CRISPR/Cas9 lentivirus according to the protocol described above. After 48 h from transduction cells were selected with 0.2 μg ml−1 of puromycin for 3 days. The selected cells were seeded as single colonies in 96-well plates by serial dilutions. After 3–4 weeks of culture, cells derived from each colony were used to assess AID-knockout by western blotting and genomic sequencing of the sgRNA target region. The genomic region flanking the CRISPR target sites was PCR amplified (Surveyor primers are listed in Supplementary Table 7), and products were purified using PCR purification kit (QIAGEN) following the manufacturer’s protocol. 400 ng total of the purified PCR products were mixed with 2 μl 10× Taq DNA Polymerase PCR buffer (Life Technologies) and ultra-pure water to a final volume of 20 μl, and subjected to a re-annealing process to enable heteroduplex formation: 95 °C for 10 min, 95 °C to 85 °C ramping at –2 °C per s, 85 °C to 25 °C at –0.25 °C per s, and 25 °C hold for 1 min. After re-annealing, products were treated with Surveyor nuclease and Surveyor enhancer S (Transgenomics) following the manufacturer’s recommended protocol, and analysed on 2% high-resolution agarose gel (Sigma Aldrich). Gels were stained with ethidium bromide (Sigma Aldrich) and imaged with a Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensities. Indel percentage was determined by the formula, 100 × (1 – (1 – (b + c) / (a + b + c)) 1 / 2), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each cleavage products. DNA was prepared from mouse and human B cells at day 4 of culture using rapid lysis buffer containing 10 μg ml−1 Proteinase K and incubation at 56 °C overnight, followed by standard isopropanol extraction, wash in ethanol 70% and resuspension in TE buffer. HTGTS libraries were generated by emulsion-mediated PCR (EM–PCR) methods as previously described9. In brief, genomic DNA was digested overnight with HaeIII frequent cutter enzyme. HaeIII-generated blunt ends were A-tailed with Klenow polymerase (3′–5′ exo-; New England Biolabs). An asymmetric adaptor (composed of an upper linker and a lower 3′-modified linker) was then ligated to fragmented DNA. To remove the unrearranged I-SceI cassettes or the unrearranged endogenous c-myc locus, ligation reactions were digested with both EcoRV and XbaI. In the first round of PCR, DNA was amplified using a biotinylated forward primer and an adaptor-specific reverse primer and Phusion polymerase (Thermo-Scientific). 20 PCR cycles were performed in the following conditions: 98 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. Multiple reactions were performed in generating large-scale libraries. Biotinylated PCR products were isolated using the Dynabeads MyOne Streptavidin C1 kit (Invitrogen), followed by an additional 2-h-digestion with blocking enzymes was performed. PCR products were eluted from the beads by 30 min incubation at 65 °C in 95% formamide/10 mM EDTA and purified. The purified products were then amplified in a second round with em-PCR in an oil-surfactant mixture. The emulsion mixture was divided into individual aliquots and PCR was performed using the following conditions: 20 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Following PCR, the products were pooled and centrifuged in a table-top centrifuge for 5 min at 14,000 r.p.m. to separate the phases and the oil layer was removed. The sample was then extracted three times with diethyl ether and DNA was re-purified. The third, non-emulsion, round of PCR (10 cycles) was performed with the same primers as in round 2, but with the addition of linkers and barcodes for Illumina Mi-seq sequencing. PCR products were size-fractionated for DNA fragments between 300 and 800 base pairs on a 1% agarose gel, column purified (QIAGEN) before loading onto Illumina Mi-seq machine for sequencing. Nucleotide sequences of junctions were generated by Mi-seq (Illumina NS500 SE150) sequencing at the Molecular Biology Core Facilities of the Dana-Farber Cancer Institute. At least three independent libraries were generated and analysed for each experimental condition (Supplementary Table 1). Oligonucleotide primers used for mouse and human libraries preparation are listed in Supplementary Table 7. First, we applied prinseq 0.2 (ref. 35) to remove sequences with exact PCR duplicates, mean quality score <20 and length <50. Next, reads for each experimental condition were demultiplexed by designed barcode, and then filtered by the presence of the primer plus additional 5 downstream bases as bait portion. Barcodes and primers used are listed in Supplementary Tables 1, 7. Lastly, the barcode, primer and bait portion of the remained sequences were masked for alignment analysis. The sequences for each experiment were aligned and filtered as previously described9. Briefly, we aligned sequences to the mouse reference genome (GRCm37/mm9) or human genome (GRCh38/hg38) using BLAT, and then filtered artificial junctions by removing PCR repeats (reads with same junction position in alignment to the reference genome and a start position in the read less than 3 bp apart), invalid alignments (including alignment scores <30, reads with multiple alignments having a score difference <4 and alignments having 10-nucleotide gaps) and ligation artefacts (for example, random HaeIII restriction sites ligated to bait breaksite). Translocation junction position was determined on the basis of the genomic position of the 5′ end of the aligned read. Translocation junctions data from similar size biological replicates were pooled for hotspots identification. First, we employed SICER 1.1 (ref. 36) to identify candidate regions where HTGTS junctions were significantly enriched against genome-wide background. The parameters used were as follows: window size, 1,000; gap size, 2,000; e-value, 0.000001; redundancy, 1; effective genome fraction, 0.77 for mouse or 0.74 for human. Next, we eliminated from analysis the following hotspots: (1) hotspots in the region ± 4 Mb around Myc bait breaksite including the Pvt1 gene as previously described9; (2) hotspots with junctions number less than 5; (3) hotspots with strand bias. We used the following entropy formula to measure strand bias as S = –P × log (P) – (1 – P) × log (1 – P), where P is the percentage of junctions from the plus stand, and 1 – P is the percentage of junctions from the minus strand. If P or 1 – P were <10% (entropy S < 0.47), we eliminated the hotspot for a strand bias; (4) hotspots without significant enrichment against the local background. The local background P value was calculated by Poisson distribution against the region that surrounds the hotspot (± 3 times the size of the hotspot). Bonferroni correction was used to adjust P value for multiple tests. We set adjusted P = 0.01 as significance level. For JeKo-1, owing to its complex karyotype37, which increases the local noise level, we set more stringent criteria for hotspot identification, including adjust P < 0.00001 and region size <30 kb. Hotspots from different experiments that partially overlapped were merged to define common hotspot regions that were used as reference to compare junction frequency between different experiments. Translocation junction frequencies in hotspots were normalized to reads per million (RPM). In box plot for fold-change comparison, to avoid ‘division by zero’ error, 0 was replaced with 1, and then normalized to corresponding RPM in library. For clustering heat map, the RPM was transformed into a log value, and then median centred. The genome-wide translocation circle plots were made using Circos tool38. Translocation junction distributions were visualized by IGV 2.3.6 (ref. 39). For translocation frequency distribution around ConvT or SE centres, centres were defined as the central bp position of the ConvT or SE region, as we previously defined10. Regions ± 4 Mb around the I-SceI c-myc breaksite on chromosome 15 and the IgH S regions on chromosome 12 were excluded in the analysis of junctions around TSS, ConvT or SE centres. For SE analysis, hotspots embedded within two adjacent SEs with centre-to-centre distance <100 kb were excluded because it was not possible to univocally assign them with one of the two SEs. All ChIP–seq data used in this study were obtained from previously published data including SE10, AID17, Spt5 and Pol II20. Statistical significance of differential junction frequency in hotspots were performed using SICER 1.1 (ref. 36) with the following parameters: window size, 1,000; gap size, 2,000; e-value, 0.000001; effective genome fraction, 0.77 (mouse) or 0.74 (human); and FDR = 0.01 or FDR = 0.1. Nuclei were isolated at day 2 from B cells activated with anti-CD40 plus IL-4 and treated with PI3K inhibitors, as previously described9. GRO-seq libraries were sequenced on the Hi-seq 2,000 platform with single-end reads and analysed as follows: GRO-seq data were aligned using Bowtie software40 mouse reference genome (GRCm37/mm9). Uniquely mapped, non-redundant sequence reads were retained. Next, we used HOMER software to count reads and calculate the nascent RNA expression levels as RPKM (reads per 1,000 bp per million mapped reads) in whole genes or in focal translocation clusters, and to identify transcripts from both strands of chromosomes41. The ConvT region was defined as sense and antisense transcription overlaps that were longer than 100 bp10. Statistical analysis for differential expression and log fold-change calculation were performed using DESeq2 (ref. 42) in whole genes or in focal translocation clusters. The MA-plot of log fold-changes against mean of normalized counts were generated by function plotMA in R package DESeq2 (ref. 42). Phusion High Fidelity DNA polymerase (Thermo-Scientific) was used to amplify selected regions from template genomic DNA. Oligonucleotide primers are listed in Supplementary Table 7: amplification conditions for each gene are available on request. Amplification products were purified using PCR purification kit (QIAGEN) and GEL extraction kit (QIAGEN) following the manufacturer’s protocol and sequenced bi-directionally in a Mi-seq (Illumina NS500) sequencing platform at the Molecular Biology Core Facilities of the Dana-Farber Cancer Institute. For SHM calculations, mouse and human intragenic and intergenic regions were targeted re-sequenced with primers indicated in Supplementary Table 7. Sequences with mean quality score <20 and length <50 were removed. Samples with less than 100 reads were excluded from analysis. The remained sequences were used to calculate mutation rate. Sequences obtained from each designed region were aligned to the reference sequence using BLASTN with alignment length >200. Mutations were calculated after filtering steps, as previously described43. Briefly, mutations first had to pass a Neighbourhood Quality Standard criteria requiring a minimum Phred score of 30 for the mutation itself, and 20 for the five adjacent bases on either side. Mutations that were within five bases of more than two additional mutations were excluded. Mutations within two bases of a deletion or insertion were also excluded. In addition, bases with mutation rate >0.01 were excluded as a result of overwhelming influence of sequence error or SNP, of which bases with mutation rate >0.2 were further regarded as SNP and were excluded. Finally, the average base mutation rate of 1– 200 bp passing the above criteria were calculated from forward sequence, as well as reverse sequences if applicable. For average base mutation rates of C-to-T or G-to-A transitions, only C or G bp sites we counted. Mutations on the VB1-8 productive allele were performed and analysed as recently described19. Source code for genomic event analysis tools (GEAT) developed in our laboratory to perform the analysis is available at https://github.com/geatools/geat. All sequencing data has been deposited in the Gene Expression Omnibus database under accession number GSE77788. Source Data for figures are provided with the online version of the paper.

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