Kiyonami R.,ThermoFisher Scientific
Methods in molecular biology (Clifton, N.J.) | Year: 2010
Proteomics is gradually shifting from pure qualitative studies (protein identification) to large-scale quantitative experiments, prompted by the growing need to analyze consistently and precisely a large set of proteins in biological samples. The selected reaction monitoring (SRM) technique is increasingly applied to quantitative proteomics because of its selectivity (two levels of mass selection), its sensitivity (non-scanning mode), and its wide dynamic range. This account describes the different steps in the design and the experimental setup of SRM experiments. Source
Thermofisher Scientific | Date: 2012-07-30
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.).
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were carried out using the TR146 buccal epithelial squamous cell carcinoma line32 obtained from the European Collection of Authenticated Cell Cultures (ECACC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were routinely tested for mycoplasma contamination using mycoplasma-specific primers and were found to be negative. Prior to stimulation, confluent TR146 cells were serum-starved overnight, and all experiments were carried out in serum-free DMEM. C. albicans wild-type strains included the autotrophic strain BWP17 + CIp30 (ref. 33) and the parental strain SC5314 (ref. 34). Other C. albicans strains used and their sources are listed in Extended Data Tables 1 and 2. C. albicans cultures were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 °C overnight. Cultures were washed in sterile PBS and adjusted to the required cell density. Antibodies to phospho-MKP1 and c-Fos were from Cell Signalling Technologies (New England Biolabs UK), mouse anti-human α-actin was from Millipore (UK), and goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunologicals (Stratech Scientific, UK). Ece1p peptides were synthesized commercially (Proteogenix (France) or Peptide Synthetics (UK). ECE1 deletion was performed as previously described35. Deletion cassettes were generated by PCR36. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17 (ref. 37), was sequentially transformed38 with the ECE1-HIS1 and ECE1-ARG4 deletion cassettes and then transformed with CIp10 (ref. 39), yielding the ece1∆/Δ deletion strain. For complementation, the ECE1 gene plus upstream and downstream intergenic regions were amplified with primers ECE1-RecF3k and ECE1-RecR and cloned into plasmid CIp10 at MluI and SalI sites. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1∆/Δ + ECE1 complemented strain. For generation of the ece1Δ/Δ + ECE1 strain, the CIp10-ECE1 was amplified with primers Pep3-F1 and Pep3-R1, digested with ClaI and re-ligated, yielding the CIp10 + ECE1 plasmid. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1Δ/Δ + ECE1 strain. All integrations were confirmed by PCR/sequencing and at least two independent isogenic transformants were created to confirm results. KEX1 deletion was performed exactly as the ECE1 deletion but using primers KEX1-FG and KEX1-RG for creating the deletion cassette. Fluorescent strains of ece1Δ/Δ and BWP17 were constructed as previously described40. Briefly, the ece1Δ/Δ and BWP17 strains were transformed with the pENO1-dTom-NATr plasmid. Primers used to clone and construct the ECE1 genes and intragenic regions are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. ECE1 promoter (primers 5′ECE1prom–NarI / 3′ECE1prom–XhoI) and terminator (5′ECE1term–SacII / 5′ECE1term–SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-pECE1-GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette38. Resistance to nourseothricin was used as selective marker and correct integration of GFP into the ECE1 locus was verified by PCR. Primers for cloning and validation are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 μl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65 °C) before subjected to 2× freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at −20 °C. Precipitated pellets were washed (2× 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at −80 °C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicentre) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 Extended Data Table 4) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the reference gene and t as the control sample. TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors41, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 μg of total protein was separated on 12% SDS–PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1,000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-ray film (Fuji film). Human α-actin was used as a loading control. DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described41, 42. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Active Motif) according to the manufacturer’s protocol. Protein concentration was determined (BCA protein quantitation kit (Perbio Science)) and 5 μg of nuclear extract was assayed in the TransAM system according to the manufacturer’s protocol. Data was expressed as fold-change in A relative to resting cells. Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analysed using Bioplex Manager 6.1 software to determine analyte concentrations. Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) activity using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions. Recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve. Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously43. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 105) were added into 1 ml serum-free DMEM, incubated for 60 min (37 °C/5% CO ) and non-adherent C. albicans cells removed by aspiration. Following washing (3× 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high-magnification fields of 200 μm × 200 μm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip. C. albicans invasion of epithelial cells was determined as described previously43. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 105), for 3 h in a humidified incubator (37 °C/5% CO ). Following washing (3× PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti-Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip. TR146 cells (105 per ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 104 cfu per ml) in DMEM and incubated for 6 h (37 °C/5% CO ). Cells were washed with PBS, fixed overnight (4 °C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 μg ml−1) for 45 min at room temperature in the dark with gentle shaking (70 r.p.m.) to stain the fungal cell wall. Epithelial cells were permeabilized with 0.1% Triton X-100 for 15 min at 37 °C in the dark, then washed and stained with 10 μg ml−1 Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 μl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase images were taken per picture. For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4 °C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in high vacuum mode. Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on two-tailed t-tests in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 – beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33 °C in E3 + PTU and used as previously described40. Briefly, 4 days post-fertilization (dpf) larvae were treated with 20 μg ml−1 dexamethasone dissolved in 0.1% DMSO 1 h before infection and thereafter. For tissue damage and neutrophil recruitment, individual AB or mpo:GFP fish (respectively) were injected into the swimbladder with 4 nl of PBS with or without 25–40 C. albicans yeast cells of ece1Δ/Δ-dTomato, ece1Δ/Δ + ECE1 + dTomato, ece1Δ/Δ + ECE1 + dTomato or BWP17-dTomato. For tissue damage, 1 nl of Sytox green (0.05 mM in 1% DMSO) was injected at 20 h post-infection into the swimbladder and fish were imaged by confocal microscopy at 24 h post-infection. For neutrophil recruitment, fish were imaged at 24 h post-injection. For synthetic peptide damage, AB or α-catenin:citrine44 fish were injected with 2 nl of peptide (9 ng or 1.25 ng per fish) or vehicle (40% DMSO or 5% DMSO) + SytoxGreen (0.05 mM in 1% DMSO) or SytoxOrange (0.5 mM in 10% DMSO) and the fish imaged by confocal microscopy 4 h later. Numbers of neutrophils and damaged cells observed were counted and tabulated for each fish. Live zebrafish imaging was carried out as previously described40. Briefly, fish were anaesthetized in Tris-buffered Tricaine (200 μg ml−1, Western Chemicals) and further immobilized in a solution of 0.4% low-melting-point agarose (LMA, Lonza) in E3 + Tricaine in a 96-well plate glass-bottom imaging dish (Greiner Bio-On). Confocal imaging was carried out using an Olympus IX-81 inverted microscope with an FV-1000 laser scanning confocal system (Olympus). Images were collected and processed using Fluoview (Olympus) and Photoshop (Adobe Systems). Panels are either a single slice for the differential interference contrast channel (DIC) with maximum projection overlays of fluorescence image channels (red-green), or maximum projection overlays of fluorescence channels. The number of slices for each maximum projection is specified in the legend of individual figures. Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King’s College London. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. Mice in the same cage were part of the same treatment. The investigators were not blinded during outcome assessment. A previously described murine model of oropharyngeal candidiasis using female BALB/c mice45 was modified to use for investigating early infection events. Briefly, mice were treated subcutaneously with 3 mg per mouse (in 200 μl PBS with 0.5% Tween 80) of cortisone acetate on days −1 and +1 post-infection. On day 0, mice were sedated for ~75 min with an intra-peritoneal injection of 110 mg per kg ketamine and 8 mg per kg xylazine, and a swab soaked in a 107 cfu per ml of C. albicans yeast culture in sterile saline was placed sublingually for 75 min. After 2 days, mice were euthanized, the tongue excised and divided longitudinally in half. One half was weighed, homogenized and cultured to derive quantitative Candida counts. The other half was processed for histopathology and immunohistochemistry. C. albicans-infected murine tongues were fixed in 10% (v/v) formal-saline before being embedded and processed in paraffin wax using standard protocols. For each tongue, 5-μm sections were prepared using a Leica RM2055 microtome and silane coated slides. Sections were dewaxed using xylene, before C. albicans and infiltrating inflammatory cells were visualized by staining using Periodic Acid-Schiff (PAS) stain and counterstaining with haematoxylin. Sections were then examined by light microscopy. Histological quantification of infection was undertaken by measuring the area of infected epithelium and expressed as a percentage relative to the entire epithelial area. TR146 epithelial cells were grown in 35-mm Petri dishes (Nunc) for 48 h before recordings at low cell density (10–30% confluence). Cells were superfused with a modified Krebs solution (120 mM NaCl, 3 mM KCl, 2.5 mM CaCl , 1.2 mM MgCl , 22.6 mM NaHCO , 11.1 mM glucose, 5 mM HEPES pH 7.4). Isolated cells were recorded at room temperature (21–23 °C) in whole cell mode using microelectrodes (5–7 MΩ) containing 90 mM potassium acetate, 20 mM KCl, 40 mM HEPES, 3 mM EGTA, 3 mM MgCl , 1 mM CaCl (free Ca2+ 40 nM), pH 7.4. Cells were voltage clamped at −60 mV using an Axopatch 200A amplifier (Axon Instruments) and current/voltage curves were generated by 1 s steps between −100 to +50 mV. Treatments were applied to the superfusate to produce the final required concentration, with vehicle controls similarly applied. Data was recorded using Clampex software (PClamp 6, Axon Instrument) and analysed with Clampfit 10. TR146 cells were grown in a 96-well plate overnight until confluent. The medium was removed and 50 μl of a Fura-2 solution (5 μl Fura-2 (Life Technologies) (2.5 mM in 50% Pluronic F-127 (Life Technologies):50% DMSO), 5 μl probenecid (Sigma) in 5 ml saline solution (NaCl (140 mM), KCl (5 mM), MgCl (1 mM), CaCl (2 mM), glucose (10 mM) and HEPES (10 mM), adjusted to pH 7.4)) was added and the plate incubated for 1 h at 37 °C/5% CO . The Fura-2 solution was replaced with 50 μl saline solution and baseline fluorescence readings (excitation 340 nm/emission 520 nm) taken for 10 min using a FlexStation 3 (Molecular Devices). Ece1 peptides were added at different concentrations and readings immediately taken for up to 3 h. The data was analysed using Softmax Pro software to determine calcium present in the cell cytosol and expressed as the ratio between excitation and emission spectra. tBLMs with 10% tethering lipids and 90% spacer lipids (T10 slides) were formed using the solvent exchange technique46, 47 according to the manufacturer’s instructions (SDx Tethered Membranes Pty Ltd, Sydney, Australia). Briefly, 8 μl of 3 mM lipid solutions in ethanol were added, incubated for 2 min and then 93.4 μl buffer (100 mM KCl, 5 mM HEPES, pH 7.0) was added. After rinsing 3× with 100 μl buffer the conductance and capacitance of the membranes were measured for 20 min before injection of Ece1 peptides at different concentrations. All experiments were performed at room temperature. Signals were measured using the tethaPod (SDx Tethered Membranes Pty, Sydney, Australia). Intercalation of Ece1 peptides into phospholipid liposomes was determined by FRET spectroscopy applied as a probe-dilution assay48. Phospholipids mixed with each 1% (mol/mol) of the donor dye NBD-phosphatidylethanolamine (NBD-PE) and of the acceptor dye rhodamine-PE, were dissolved in chloroform, dried, solubilized in 1 ml buffer (100 mM KCl, 5 mM HEPES, pH 7.0) by vortexing, sonicated with a titan tip (30 W, Branson sonifier, cell disruptor B15), and subjected to three cycles of heating to 60 °C and cooling down to 4 °C, each for 30 min. Lipid samples were stored at 4 °C for at least 12 h before use. Ece1 peptide was added to liposomes and intercalation was monitored as the increase of the quotient between the donor fluorescence intensity I at 531 nm and the acceptor intensity I at 593 nm (FRET signal) independent of time. CD measurements were performed using a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co., Japan), calibrated as described previously49. CD spectra represent the average of four scans obtained by collecting data at 1 nm intervals with a bandwidth of 2 nm. The measurements were performed in 100 mM KCl, 5 mM HEPES, pH 7.0 at 25 °C and 40 °C in a 1.0 mm quartz cuvette. The Ece1-III concentration was 15 μM. Planar lipid bilayers were prepared using the Montal-Mueller technique50 as described previously51. All measurements were performed in 5 mM HEPES, 100 mM KCl, pH 7.0 (specific electrical conductivity 17.2 mS per cm) at 37 °C. Candida strains were cultured for 18 h in hyphae inducing conditions (YNB medium containing 2% sucrose, 75 mM MOPSO buffer pH 7.2, 5 mM N-acetyl-d-glucosamine, 37 °C). Hyphal supernatants were collected by filtering through a 0.2 μm PES filter, and peptides were enriched by solid phase extraction (SPE) using first C4 and subsequently C18 columns on the C4 flowthrough. After drying in a vacuum centrifuge, samples were resolubilized in loading solution (0.2% formic acid in 71:27:2 ACN/H O/DMSO (v/v/v)) and filtered through a 10 kDa MWCO filter. The filtrate was transferred into HPLC vials and injected into the LC-MS/MS system. LC-MS/MS analysis was carried out on an Ultimate 3000 nano RSLC system coupled to a QExactive Plus mass spectrometer (ThermoFisher Scientific). Peptide separation was performed based on a direct injection setup without peptide trapping using an Accucore C4 column as stationary phase and a column oven temperature of 50 °C. The binary mobile phase consisting of A) 0.2% (v/v) formic acid in 95:5 H O/DMSO (v/v) and B) 0.2% (v/v) formic acid in 85:10:5 ACN/H O/DMSO (v/v/v) was applied for a 60 min gradient elution: 0–1.5 min at 60% B, 35–45 min at 96% B, 45.1–60 min at 60% B. The Nanospray Flex Ion Source (ThermoFisher Scientific) provided with a stainless steel emitter was used to generate positively charged ions at 2.2 kV spray voltage. Precursor ions were measured in full scan mode within a mass range of m/z 300–1600 at a resolution of 70k FWHM using a maximum injection time of 120 ms and an automatic gain control target of 1e6. For data-dependent acquisition, up to 10 most abundant precursor ions per scan cycle with an assigned charge state of z = 2–6 were selected in the quadrupole for further fragmentation using an isolation width of m/z 2.0. Fragment ions were generated in the HCD cell at a normalized collision energy of 30 V using nitrogen gas. Dynamic exclusion of precursor ions was set to 20 s. Fragment ions were monitored at a resolution of 17.5k (FWHM) using a maximum injection time of 120 ms and an AGC target of 2e5. Thermo raw files were processed by the Proteome Discoverer (PD) software v220.127.116.118 (Thermo). Tandem mass spectra were searched against the Candida Genome Database (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz; status: 2015/05/03) using the Sequest HT search algorithm. Mass spectra were searched for both unspecific cleavages (no enzyme) and tryptic peptides with up to 4 missed cleavages. The precursor mass tolerance was set to 10 p.p.m. and the fragment mass tolerance to 0.02 Da. Target Decoy PSM Validator node and a reverse decoy database was used for (q value) validation of the peptide spectral matches (PSMs) using a strict target false discovery (FDR) rate of <1%. Furthermore, we used the score versus charge state function of the Sequest engine to filter out insignificant peptide hits (xcorr of 2.0 for z = 2, 2.25 for z = 3, 2.5 for z = 4, 2.75 for z = 5, 3.0 for z = 6). At least two unique peptides per protein were required for positive protein hits. TransAM and patch clamp data were analysed using a paired t-test while cytokines, LDH and calcium influx data were analysed using one-way ANOVA with all compared groups passing an equal variance test. Murine in vivo data was analysed using the Mann–Whitney test. Zebrafish data was analysed using the Kruskal–Wallis test with Dunn’s multiple comparison correction. In all cases, P < 0.05 was taken to be significant.
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.