News Article | February 15, 2017
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 | November 23, 2016
Plasmids containing the 9-kb mouse villin promoter (pBS-Villin)26, 27 and simian diphtheria toxin receptor (HBEGF (‘DTR’)) with the enhanced green fluorescent protein (pDTR–eGFP) fusion gene28 have been previously described. The pDTR–eGFP was PCR amplified with primers harbouring a 5′ BsiWI site and a 3′ MluI site and cloned into pBS-Villin. The pBS-Villin/DTR–eGFP plasmid was verified by sequencing and the transgene was isolated from the plasmid by restriction enzyme digestion and gel purification. The transgene was microinjected into fertilized eggs from C57BL/6J mice (Jackson Laboratory) and transferred into oviducts of ICR foster mothers as previously described26. Identification of the transgenic mice was performed by PCR amplification using the following primers: 5′-ACTGCTCTCACATGCCTTCT-3′ and 5′-CTTCTTCCCTAGTCCCTTGC-3′. For diphtheria toxin administration, mice were injected intraperitoneally with 2 or 10 ng g−1 diphtheria toxin (EMD Chemicals) and humanely killed 1–24 h later29. Control mice were injected with PBS. For dextran sulphate solution (DSS) (MP Biomedicals) studies, mice were supplemented with 3% DSS in the drinking water for five days. On day three, water bottles were refilled with 3% DSS solution and on day five, replaced with fresh drinking water. Mice were weighed and monitored daily for signs of distress, morbidity or mortality during the course of the experiment until they were killed on day 7. Both male and female mice ages 6–8 weeks were used for all studies. All experiments were approved by the institutional animal care and use committee and carried out in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication 86–23, revised 1985). Before isolating professional phagocytes (‘phagocytes’), VDTR and VDTR negative littermate controls were intraperitoneally injected with PBS (vehicle) or diphtheria toxin (EMD Chemicals) at a low (2 ng g−1) or high (10 ng g−1) dose per body weight. Mice were then killed 1–24 h later and phagocytes were isolated from the SILP as previously described with some modifications30. In brief, the small intestine, including the duodenum, jejunum and ileum, was excised and Peyer’s patches removed. Next, the small intestine was opened longitudinally with surgical scissors and flushed with ice-cold PBS to remove the faecal content. Intestines were then cut into 0.5-cm pieces and transferred into 50-ml conical tubes containing 20 ml of PBS. Samples were then vigorously shaken for 30 s using the vortex genie (Scientific Industries) and passed over 100-μm nylon cell strainers (BD Falcon). Fresh PBS was added to the tissue samples and the shaking and filtering process was repeated a total of eight times. To isolate and remove the intestinal epithelial cell layer, samples were washed with 20 ml of warm PBS containing 3 mM EDTA and passed over cell strainers. This was repeated three times. Flow-through was kept as purified for IECs, whereas whole tissues were further processed to isolate dendritic cell and macrophage subsets. Next, samples were washed with ice-cold PBS followed by RPMI 1640 (Sigma) containing 5% FBS to remove the EDTA. Samples were then re-suspended with RPMI 1640 containing 5% FBS, 1 mg ml−1 collagenase D (Roche), and 1 mg ml−1 DNase I (Roche) and incubated in a 37 °C water bath for 60 min. Samples were shaken every 20 min during this time. At the completion of the incubation, samples were washed with FACS buffer to remove the collagenase and then passed through an 18-gauge needle followed by a 21-gauge needle to create a single-cell suspension. Phagocytes were then enriched from samples by using a 1.065 g ml−1 OptiPrep (Sigma) density gradient according to the manufacturer’s protocol. Following centrifugation, phagocytes were isolated from both low- and mid-density bands and finally re-suspended in FACS buffer for flow cytometric analyses. Mouse spleen was digested in parallel with small intestine samples and used for single-colour compensation controls. All samples were pretreated with Fc block for 10 min at 4 °C followed by fluorescently conjugated antibody labelling at 4 °C for 60 min. The following antibodies were used for these studies: Antibodies from BioLegend including Alexa Fluor 647- or 700-conjugated anti-CD11c (clone N418), PerCP/Cy5.5-conjugated anti-CD24 (clone M1/69), APC/Cy7-conjugated anti-CD45 (clone 30-F11), APC-conjugated anti-CD64 (clone X54-5/7.1), APC-conjugated anti-CD274 (clone 10F.9G2), PerCP/Cy5.5-conjugated anti-F4/80 (clone BM8), Alexa Fluor 700-conjugated anti-Ly-6c (clone HK1.4), and Phycoerythrin (PE) or Brilliant Violet 421-conjugated anti-MHCII I-A/I-E (clone M5/114.15.2); antibodies from eBioscience including FITC-conjugated anti-CD4 (clone RM4-5), PE/Cy7-conjugated anti-CD11b (clone M1/70) and PE-conjugated anti-CD103 (clone 2E7); and TxRed-conjugated anti-CD45 (clone 30-F11) from Invitrogen. Live/Dead Aqua (Life Technologies) was used to discriminate viable cells. Phagocytes isolated from the SILP were surface stained for: APC/Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD11c, Brilliant Violet 421-conjugated anti-MHCII I-A/I-E, PE/Cy7-conjugated anti-CD11b, PE-conjugated anti-CD103, PerCP/Cy5.5-conjugated anti-CD24, and APC-conjugated anti-CD64. The identification of phagocytes from VDTR mice with IEC cargo was determined by the presence of eGFP and this gate was defined on the basis of C57BL/6J and VDTR− littermate controls that were eGFP−. Sample acquisition was performed using the LSRFortessa (BD Biosciences) and data analyses were performed using the FlowJo analytical software (Tree Star). To sort phagocytes with and without apoptotic IEC cargo, the following surface markers were used: APC/Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD11c, Brilliant Violet 421-conjugated anti-MHCII I-A/I-E, PE/Cy7-conjugated anti-CD11b, PE-conjugated anti-CD103, PerCP/Cy5.5-conjugated anti-CD24, and APC-conjugated anti-CD64. The identification of phagocytes from VDTR mice with IEC cargo was determined by the presence of eGFP and this gate was defined on the basis of C57BL/6J and VDTR− littermate controls that were eGFP−. Sorted populations were live, CD45+MHCII+CD11c+ phagocytes that were either eGFP− or eGFP+ including (i) CD103+CD11b−CD24+CD64− (hereafter CD103), (ii) CD103+CD11b+ CD24−CD64+ (hereafter CD103 CD11b), and (iii) CD103−CD11b+CD24− CD64+ (hereafter CD11b) for a total of six populations. Owing to the four-sample sort-maximum of the instrument, the three eGFP+ populations were collected first and then fresh collection tubes were added for the three eGFP− populations. Cells were sorted directly into 0.5 ml TRIzol LS reagent (Life Technologies) for microarray processing (see below). Each sort was performed at 4 h following diphtheria toxin administration and consisted of 3–4 pooled VDTR mice. The following are the cell yield ranges for each subset: 1,000–5,000 eGFP+CD103+; 3,000–9,000 eGFP+CD103+CD11b+; 10,000–40,000 eGFP+CD11b+; 4,500–10,000 eGFP−CD103+; 40,000–80,000 eGFP−CD103+CD11b+; and 30,000–100,000 eGFP−CD11b+. FACS was conducted on the FACSAria IIu SORP (BD Biosciences). The following are the RNA yield ranges for each subset: 200–2,400 pg eGFP+CD103+; 200–3,000 pg eGFP+CD103+CD11b+; 600–3,000 pg eGFP+CD11b+; 200–4,600 pg eGFP−CD103+; 600–5000 pg eGFP−CD103+CD11b+; and 450–4,000 pg eGFP−CD11b+. The purity and identity of each subset was validated as indicated in Extended Data Fig. 5 and according to markers as previously reported31. For analysis of IEC engulfment by CD11c+ phagocytes, single-cell suspensions were prepared as described for flow cytometric analyses and acquired using the IS 100 Imaging flow cytometer (Amnis Corp). Phagocytes with eGFP+ cargo were identified as those that contained single nuclei and were CD45+, CD11c+ and MHCII+. Data were analysed using IDEAS software (Amnis Corp) and spectrally compensated using a compensation matrix generated from the following single-colour controls; FITC-conjugated CD4, PE-conjugated MHCII, Alexa Fluor647-conjugated CD11c, TxRed-conjugated CD45, and Hoechst stain. Total RNA was isolated from mouse small intestine using RNeasy mini-kit (Qiagen) and quantified by a spectrophotometer. Reverse transcription was performed with Superscript III (Invitrogen) and cDNA was synthesized using the Mastercycler ep (Eppendorf). Real-time quantitative RT–PCR was conducted in duplicate on a ViiA 7 Real-time PCR System (Life Technologies) using TaqMan quantitative PCR Master Mix at a concentration of 1× (Applied Biosystems) or SYBR Green Real-Time PCR Master Mixes for the eGFP and HBEGF (‘DTR’) transgenes. Samples were normalized to β-actin and relative expression was determined by 2-ΔΔC method. Forward (FW) and reverse (RV) primers for SYBR Green include: All probe sequences are in the format: 5′ FAM-sequence-BHQ-1 3′ and together with forward (FW) and reverse (RV) primer pairs were synthesized by Biosearch Technologies. 5′-AGCCACCCCCACTCCTAAGAGGAGG-3′ Actb probe, 5′-GAAGTCCCTCACCCTCCCAA-3′ Actb FW, 5′-GGCATGGACGCGACCA-3′ Actb RV; 5′-AAATCGGTGATCCAGGGATTGTTCCA-3′ Acadsb probe, 5′-CCTCTGGTTTCCTCTATGGATGA-3′, Acadsb FW, 5′-TCCCTCCATATTGTGCTTCAAC-3′ Acadsb RV; 5′-CGGGACAGGGCAACTCTTGCAA-3′ Aldh1a2 probe, 5′-GCTTGCAGACTTGGTGGAA-3′ Aldh1a2 FW, 5′-GCTTGCAGGAATGGCTTACC-3′ Aldh1a2 RV; 5′-CCCACTTTCCTTGTGGTACTCTGGAC-3′ Alox5ap probe, 5′-CAACCAGAACTGCGTAGATGC-3′ Alox5ap FW, 5′-GAAGGCGGCAGGGACTTG-3′ Alox5ap RV; 5′-TGCCTTTAGTGGCCTCATTGTTCC-3′ Atrn probe, 5′-GGACTCAATCTACGCACCTCTGAT-3′ Atrn FW, 5′-GCCGTCTCATTGCCATCTCTT-3′ Atrn RV; 5′-TTGGCATCAATCTGAGCTGTTGGTG-3′ Axl probe, 5′-GCCCATCAACTTCGGAAGAAAG-3′ Axl FW, 5′-CCTCTGGCACCTGTGATATTCC-3′ Axl RV; 5′-AGTGAAGGAGTTCTTCTGGACCTCAA-3′ Ccl22 probe, 5′-CACCCTCTGCCATCACGTT-3′ Ccl22 FW, 5′-ATCTCGGTTCTTGACGGTTATCA-3′ Ccl22 RV; 5′-CCACTGCTCATGGATATGTTGAACAATAGAGACC-3′ Ccr2 probe, 5′-AGGGTCACAGGATTAGGAAGGTT-3′ Ccr2 FW, 5′-CGTTCTGGGCACCTGATTTAA-3′ Ccr2 RV; 5′-CAGTGCCCAAGTGGAGGCCTTGATC-3′ Ccr7 probe, 5′-CACGCTGAGATGCTCACTGG-3′ Ccr7 FW, 5′-ATCTGGGCCACTTGGATGG-3′ Ccr7 RV; 5′-AGATTCGCTGTCACCAGCACAGACA-3′ Cd40 probe, 5′-TCTCAGCCCAGTGGAACA-3′ Cd40 FW, 5′-CGGTGCCCTCCTTCTTAACC-3′ Cd40 RV; 5′-CGAATCACGCTGAAAGTCAATGCCC-3′ Cd274 probe, 5′-CGGTGGTGCGGACTACAAG-3′ Cd274 FW, 5′-CCCTCGGCCTGACATATTAGTTC-3′ Cd274 RV; 5′-TTCCCAGGGCTTGAGGCTCCC-3′ Cd300a probe, 5′-GGCCACCGTGAACATGACTA-3′ Cd300a FW, 5′-GCAGGAGAGCTAACACAGACAAC-3′ Cd300a RV; 5′-ATGGAAAATGGGTGGCGTCTAACCCA-3′ Cfh probe, 5′-CCGAACACTTGGCACTATTGTAA-3′ Cfh FW, 5′-CTCCGGGATGCCCACAAG-3′ Cfh RV; 5′-CCCTGAACAACCAACAGATGACACTGG-3′ Elf3 probe, 5′-GGCACTGAAGACTTGGTGTTG-3′ Elf3 FW, 5′-CCCTGAACAACCAACAGATGACACTGG-3′ Elf3 RV; 5′-AGCTGACAGATACACTCCAAGCGGA-3′ Fos probe, 5′-AGTGCCGGAATCGGAGGA-3′ Fos FW, 5′-TGCAACGCAGACTTCTCATC-3′ Fos RV; 5′-CTGCTCCTGCTGGCTTCCGAGT-3′ Gas6 probe, 5′-CTGGGCACTGCGCTTCTG-3′ Gas6 FW, 5′-CGCAACAGCACAGTGTGA-3′ Gas6 RV; 5′-TCTTATGCAGACTGTGTCCTGGCA-3′ Ido1 probe, 5′-GGGCCTGCCTCCTATTCTG-3′ Ido1 FW, 5′-CCCACCAGGAAATGAGAACAGA-3′ Ido1 RV; 5′-TCACAAGCAGAGCACAAGCCTGTC-3′ Il1b probe, 5′-AAAGACGGCACACCCACCCTGC-3′ Il1b FW, 5′-TGTCCTGACCACTGTTGTTTCCCAG-3′ Il1b RV; 5′-TCTGCAAGAGACTTCCATCCAGTTGCCT-3′ Il6 probe, 5′-CCAGAAACCGCTATGAAGTTCC-3′ Il6 FW, 5′-TCACCAGCATCAGTCCCAAG-3′ Il6 RV; 5′-TTCAAACAAAGGACCAGCTGGACA-3′ Il10 probe, 5′-TCAGCCAGGTGAAGACTTTC-3′ Il10 FW, 5′-GGCAACCCAAGTAACCCTTA-3′ Il10 RV; 5′-TAACTGGGATCCAGGCACGCC-3′ Ly75 probe, 5′-GTCAGACTTCAGGCCACTCAA-3′ Ly75 FW, 5′-TGACCCACCAATCACAGGT-3′ Ly75 RV; 5′-TCCCTTACTTTATTAAGCAGCCTGAGAGTG-3′ Mertk probe, 5′-TGATCCCATATACGTGGAAGTTCA-3′ Mertk FW, 5′-CCTGGCAGGTGAGGTTGAAG-3′ Mertk RV; 5′-TTTGCGTCTGACTGCCGAGACTC-3′ Muc2 probe, 5′-CCTGGCCTCTGTGATTACAAC-3′ Muc2 FW, 5′-GGTGCACAGCAAATTCCTTGTAG-3′ Muc2 RV; 5′-TCGCAACCAGATCGGAGATGTGG-3′ Nlrc5 probe, 5′-CCAGAACTCAGGAAATTTGACTTGA-3′ Nlrc5 FW, 5′-TTTGGCAAGATGGCAGCTAA-3′ Nlrc5 RV; 5′-CTGCTGCCTCACTTCTAGCTTCTGC-3′ Nlrp3 probe, 5′-GTTGCCTGTTCTTCCAGACT-3′ Nlrp3 FW, 5′-GGCTCCGGTTGGTGCTTAG-3′ Nlrp3 RV; 5′-TAGGCTGCTTTGGGAATGGCACC-3′ Oasl1 probe, 5′-CGCGTGCTCAAGGTACTCAAG-3′ Oasl1 FW, 5′-GACCAGCTCCACGTCTGTAG-3′ Oasl1 RV; 5′-TTGTGATGACTACATGGTCACACTCTTC-3′ Plac8 probe, 5′-GAACCCGATACGGCATTCCT-3′ Plac8 FW, 5′-TCTTGCCATCCAGCTCCTTAG-3′ Plac8 RV; 5′-ACCAACACATCGGAGCTGCGGA-3′ Relb probe, 5′-GAGCCTGTCTACGACAAGAAGTC-3′ Relb FW, 5′-GCCCGCTCTCCTTGTTGATTC-3′ Relb RV; 5′-AGTTATGCACGAGTGCGAGCTGT-3′ Spred1 probe, 5′-CGGCGACTTCTGACAACGATA-3′ Spred1 FW, 5′-GGTAGCCATCCACCACTTGAG-3′ Spred1 RV; 5′-AGAGGTCACCCGCGTGCTAATGGTG-3′ Tgfb1 probe, 5′-CCCGAAGCGGACTACTATGC-3′ Tgfb1 FW, 5′-ATAGATGGCGTTGTTGCGGT-3′ Tgfb1 RV; 5′-CTCTGCCTGCATCCAATCACTCTCA-3′ Timd4 probe, 5′-GGTCCGCCTTCACTACAGAATC-3′ Timd4 FW, 5′-GGCCTGAGTACGGCTATGTC-3′ Timd4 RV; 5′-TGGGCTTTCCGAATTCACTGGAGC-3′ Tnf probe, 5′-ATGCACCACCATCAAGGACTCAA-3′ Tnf FW, 5′-ACCACTCTCCCTTTGCAGAACTC-3′ Tnf RV; 5′-TCAACTGGTGTCGTGAAGTCAGGA-3′ Tnfaip3 probe, 5′-TCCCTGGAAAGCCAGAAGAAG-3′ Tnfaip3 FW, 5′-GAGGCAGTTTCCATCACCATTG-3′ Tnfaip3 RV; 5′-TCCGGAGCTACTTCAAGCAAGGC-3′ Vil1 probe, 5′-GGCAACGAGAGCGAGACTT-3′ Vil1 FW, 5′-CGCTGGACATCACAGGAGTT-3′ Vil1 RW. A total of five sorting experiments with a pool of 3–4 mice were performed for the cDNA microarrays. Following cell sorting into TRIzol LS reagent, samples were shipped on dry ice to the Center for Functional Genomics and the Microarray & HT Sequencing Core Facility at the University at Albany (Rensselaer). A sample clean-up step was performed using RNeasy columns (Qiagen) that included DNase treatment. The isolated RNA was checked for quality using NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent), following which 1 ng of total RNA was processed using WT-Ovation Pico RNA Amplification System (NuGEN). A total of three biological replicates were used for the microarray. When required, RNA was pooled from additional sorts to achieve the 1 ng of total RNA needed for the amplification system. The following are the sort experiments used for each sample: (2, 2 and 5, 2 and 5) eGFP+CD103+; (2, 3, 2 and 5) eGFP+CD103+CD11b+; (2, 4, 5) eGFP+CD11b+; (3, 2 and 4 and 5, 2 and 4 and 5) eGFP−CD103+; (2, 3, 4) eGFP−CD103+CD11b+; and (2, 3, 5) eGFP−CD11b+. RNA was reverse-transcribed and sense-target cDNAs were targeted using the standard NuGEN protocol and hybridized to Affymetrix mouse Gene 2.0 ST arrays. These arrays were then washed, stained on a FS 450 station, and scanned on a GeneChip 3000 7G scanner using Affymetric GeneChip Command Console Software (AGCC). The Affymetrix microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO)32 and are accessible with the GEO series accession number GSE85682. Fold changes and statistical significance were identified as those genes that were differentially expressed between eGFP+ and eGFP− subsets by at least 1.2 fold (ANOVA (Benjamini–Hochberg false discovery rate correction Q < 0.05) and Tukey’s HSD post-hoc test (P < 0.05; -1.2> fold >1.2) and determined using R software (version 3.2.0). Hierarchical clustering of differentially expressed genes meeting the aforementioned criteria were Z-scored and plotted with heatmap.2 (gplots version 2.17.0, CRAN/R). Principal component analyses of the 1,534 genes (ANOVA (Benjamini–Hochberg false discovery rate correction Q < 0.05); 4.8% of total) with the most variable expression in each CD11c+ subset with and without eGFP cargo were generated using R software which are freely available online. Small and large intestine were dissected and fixed in 10% formalin (Fisher Scientific) for 24 h and then processed for paraffin embedding. Tissue blocks were then cut into 5-mm sections, de-paraffinized by xylene immersion, and hydrated by serial immersion in 100%, 90%, 80%, 70% ethanol and PBS. Antigen retrieval was performed by heating samples in a pressure cooker (Cuisinart) in citrate buffer solution (10 mM citric acid monohydrate, 0.05% Tween 20 and PBS). Sections were then washed twice in PBS, blocked for 30 min in blocking buffer (10% BSA, 0.3% Triton X-100 (Sigma) and TBS), and prepared for labelling. TdT-mediated dUTP nick end labelling (TUNEL) was performed using the in situ cell-death detection kit, TMR red (Roche), per the manufacturer’s instructions, stained with DAPI, and mounted using Fluoromount-G (Southern Biotech). For cleaved caspase-3 (Cell Signaling), samples were labelled for 60 min at room temperature, stained with DAPI, and mounted using Fluoromount-G. For paraffin images, eGFP signal was not present owing to sample quenching following paraffin embedding and processing. Small and large intestine were dissected and fixed overnight in 1.6% paraformaldehyde (Thermo Scientific) containing 20% sucrose at 4 °C. Samples were then placed in OCT (Tissue-Tek) and snap-frozen over dry ice. Tissue sections of 8-mm thickness were cut, air-dried and blocked using blocking solution. Tissues were then labelled using an Alexa Fluor 594-conjugated phalloidin (Invitrogen) or a primary mouse anti-mouse pan-cytokeratin antibody (clone PCK-26) (Abcam) for 60 min in a humidified atmosphere followed by a secondary goat anti-mouse Alexa Fluor 594 (Thermo Fisher Scientific) for 30 min, then stained with DAPI, and mounted using Fluoromount-G. For fluorescent in situ hybridization, small intestine and large intestine were dissected and prepared as described for frozen sections33. Following tissue blocking, sections were incubated with 0.45 pmol μl−1 eubacterial oligonucleotide probe (AminoC6 + Alexa Fluor 594) 5′-GCTGCCTCCCGTAGGAGT-3′; (Operon)33 in a pre-chilled hybridization buffer (Sigma) overnight at 4 °C. Sections were counterstained with DAPI and mounted with Fluoromount-G. To label small intestine tissues, the whole-mount histology protocol was modified from previously described methods34. In brief, small intestine samples were excised, opened longitudinally, and washed in ice-cold PBS. Samples were then cut to 1 cm in length and placed in 6-ml polypropylene tubes (BD Biosciences). Next, samples were incubated with Fc block at 10 μg ml−1 in 200 μl of 2% paraformaldehyde with 1% FBS, 0.3% Triton X-100 in PBS for 3 h at 4 °C with gentle rocking. After blocking and fixing, samples were put into new polypropylene tubes and labelled using 3 μg ml−1 of the following antibodies: PE-conjugated anti-CD11c (clone N418) (eBioscience), APC-conjugated anti-CD31 (clone 390) (eBioscience) and anti-cleaved caspase-3 at 1:100. All labelling was conducted in the dark at 4 °C with gentle rocking for 3 h. Finally, samples were washed for 30 min in the dark at 4 °C with fresh PBS and mounted for imaging. Conventional microscopy was performed using the Eclipse Ni-E motorized upright microscope (Nikon) and images were acquired from paraffin, frozen, and whole mount tissue sections using a Nikon DS-Qi1 Mc camera. Cell quantification was calculated using NIS Elements imaging software (Nikon) and the object count application including intensity of stain thresholds and area restriction filters. Confocal microscopy was performed at the Microscope CORE at the Icahn School of Medicine at Mount Sinai using the Leica SP5 DM upright microscope and Leica LAS AF software. Naive mouse splenic CD4+ T cells were isolated by sorting with MACS CD4+ beads (Miltenyi Biotech) according to the manufacturer’s instructions and then by FACS using the FACSAria IIu SORP. T cells were sorted on the basis of the following criteria: live, CD45+CD3+CD4+ CD25−CD44−/lowCD62L+/high. Surface antibodies for sorting included: APC/Cy7-conjugated anti-CD45, eFluor 450-conjugated anti-CD3 (clone 145-2c11), PE-conjugated anti-CD4, APC-conjugated anti-CD25 (clone PC61.5), FITC-conjugated CD62L (clone MEL-14), and Alexa 700-conjugated anti-CD44 (clone IM7) (all eBiosciences). 1 × 105 T cells were then cultured with 1 × 104 eGFP+ or eGFP− CD103 dendritic cells sorted from the MLN which were identified as: live, CD45+MHCIIhiCD11c+, eGFP+ or eGFP−, CD103+CD11b− using the aforementioned antibodies for flow cytometry. These cells were cultured in round-bottom 96-well plates (Falcon) with complete IMDM (Gibco) supplemented with 10% FBS, 100 μg ml−1 penicillin, 100 μg ml−1 streptomycin, 2 mM l-glutamine, 10 mM HEPES and 1 nM sodium pyruvate for 5 days. Additionally, all cultures were supplemented with 1 μg ml−1 of soluble anti-CD3 (clone 2C11) as well as 5 ng ml−1 of recombinant human anti-IL-2 (Pepro Tech) on days 2 and 4. A total of 2 ng ml−1 of recombinant human anti-TGFβ1 (clone 1D11 R&D systems) was added to culture wells where indicated on days 1 and 4. On day 5, cells were first surface stained with FITC-conjugated anti-CD25, PE/Cy7-conjugated anti-CD4, Alexa Fluor 700-conjugated anti-CD3, and APC/Cy7-conjugated anti-CD45, followed by fixation and permeabilization (using the concentrate and diluent provided by eBioscience), and finally intracellular staining for eFluor 450-conjugated anti-FOXP3 (clone FJK-16 s), PE-conjugated anti-RORγ(t) (clone B2D), PerCP-eFluor 710-conjugated anti-GATA-3 (clone TWAJ), and APC-conjugated anti-T-bet (clone eBio4B10) (all eBioscience). Data are presented as mean ± s.e.m. Statistical significances were determined by a one-way ANOVA with Dunnett’s and Newman–Keuls post-tests or unpaired two-tailed t-test with Welch correction where specified. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not statistically significant (P > 0.05). 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. The Affymetrix microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under GEO series accession number GSE85682.
News Article | November 9, 2016
The Global Cell Surface Markers Identification Market Research Report 2016 is a valuable source of insightful data for business strategists. It provides the Cell Surface Markers Identification industry overview with growth analysis and historical & futuristic cost, revenue, demand and supply data (as applicable). The research analysts provide an elaborate description of the value chain and its distributor analysis. This Cell Surface Markers Identification market study provides comprehensive data which enhances the understanding, scope and application of this report. This report provides comprehensive analysis of lKey market segments and sub-segments lEvolving market trends and dynamics lChanging supply and demand scenarios lQuantifying market opportunities through market sizing and market forecasting lTracking current trends/opportunities/challenges lCompetitive insights lOpportunity mapping in terms of technological breakthroughs The Major players reported in the market include: Abbott BD Biosciences Beckman Coulter Bio-Rad CellaVision AB Thermo Fisher Scientific, Inc. EMD Millipore Horiba Ltd. Mindray Medical International Limited Ortho-Clinical Diagnostics, Inc. QIAGEN N.V. Siemens Healthcare Grifols, S.A Dako Denmark A/S eBioscience, Inc. Sysmex Corporation F. Hoffmann-La Roche Ltd. ... Reasons for Buying this Report This report provides pin-point analysis for changing competitive dynamics It provides a forward looking perspective on different factors driving or restraining market growth It provides a six-year forecast assessed on the basis of how the market is predicted to grow It helps in understanding the key product segments and their future It provides pin point analysis of changing competition dynamics and keeps you ahead of competitors It helps in making informed business decisions by having complete insights of market and by making in-depth analysis of market segments It provides distinctive graphics and exemplified SWOT analysis of major market segments Global Cell Surface Markers Identification Market Research Report 2016 Chapter 1 Cell Surface Markers Identification Market Overview 1.1 Product Overview and Scope of Cell Surface Markers Identification 1.2 Cell Surface Markers Identification Market Segmentation by Type 1.2.1 Global Production Market Share of Cell Surface Markers Identification by Type in 2015 1.2.1 Type I 1.2.2 Type II 1.2.3 Type III 1.3 Cell Surface Markers Identification Market Segmentation by Application 1.3.1 Cell Surface Markers Identification Consumption Market Share by Application in 2015 1.3.2 Application I 1.3.3 Application II 1.3.4 Application III 1.4 Cell Surface Markers Identification Market Segmentation by Regions 1.4.1 North America 1.4.2 China 1.4.3 Europe 1.4.4 Southeast Asia 1.4.5 Japan 1.4.6 India 1.5 Global Market Size (Value) of Cell Surface Markers Identification (2011-2021) Chapter 2 Global Economic Impact on Cell Surface Markers Identification Industry 2.1 Global Macroeconomic Environment Analysis 2.1.1 Global Macroeconomic Analysis 2.1.2 Global Macroeconomic Environment Development Trend 2.2 Global Macroeconomic Environment Analysis by Regions 2.3 Effects to Cell Surface Markers Identification Industry Chapter 3 Global Cell Surface Markers Identification Market Competition by Manufacturers 3.1 Global Cell Surface Markers Identification Production and Share by Manufacturers (2015 and 2016) 3.2 Global Cell Surface Markers Identification Revenue and Share by Manufacturers (2015 and 2016) 3.3 Global Cell Surface Markers Identification Average Price by Manufacturers (2015 and 2016) 3.4 Manufacturers Cell Surface Markers Identification Manufacturing Base Distribution, Sales Area and Product Type 3.5 Cell Surface Markers Identification Market Competitive Situation and Trends 3.5.1 Cell Surface Markers Identification Market Concentration Rate 3.5.2 Cell Surface Markers Identification Market Share of Top 3 and Top 5 Manufacturers 3.5.3 Mergers & Acquisitions, Expansion Chapter 4 Global Cell Surface Markers Identification Production, Revenue (Value) by Region (2011-2016) 4.1 Global Cell Surface Markers Identification Production by Region (2011-2016) 4.2 Global Cell Surface Markers Identification Production Market Share by Region (2011-2016) 4.3 Global Cell Surface Markers Identification Revenue (Value) and Market Share by Region (2011-2016) 4.4 Global Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.5 North America Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.6 Europe Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.7 China Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.8 Japan Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.9 Southeast Asia Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) 4.10 India Cell Surface Markers Identification Production, Revenue, Price and Gross Margin (2011-2016) Chapter 5 Global Cell Surface Markers Identification Supply (Production), Consumption, Export, Import by Regions (2011-2016) 5.1 Global Cell Surface Markers Identification Consumption by Regions (2011-2016) 5.2 North America Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) 5.3 Europe Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) 5.4 China Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) 5.5 Japan Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) 5.6 Southeast Asia Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) 5.7 India Cell Surface Markers Identification Production, Consumption, Export, Import by Regions (2011-2016) Chapter 6 Global Cell Surface Markers Identification Production, Revenue (Value), Price Trend by Type 6.1 Global Cell Surface Markers Identification Production and Market Share by Type (2011-2016) 6.2 Global Cell Surface Markers Identification Revenue and Market Share by Type (2011-2016) 6.3 Global Cell Surface Markers Identification Price by Type (2011-2016) 6.4 Global Cell Surface Markers Identification Production Growth by Type (2011-2016) Chapter 7 Global Cell Surface Markers Identification Market Analysis by Application 7.1 Global Cell Surface Markers Identification Consumption and Market Share by Application (2011-2016) 7.2 Global Cell Surface Markers Identification Consumption Growth Rate by Application (2011-2016) 7.3 Market Drivers and Opportunities 7.3.1 Potential Applications 7.3.2 Emerging Markets/Countries Chapter 8 Global Cell Surface Markers Identification Manufacturers Analysis 8.1 Abbott 8.1.1 Company Basic Information, Manufacturing Base and Competitors 8.1.2 Product Type, Application and Specification 8.1.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.1.4 Business Overview 8.2 BD Biosciences 8.2.1 Company Basic Information, Manufacturing Base and Competitors 8.2.2 Product Type, Application and Specification 8.2.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.2.4 Business Overview 8.3 Beckman Coulter 8.3.1 Company Basic Information, Manufacturing Base and Competitors 8.3.2 Product Type, Application and Specification 8.3.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.3.4 Business Overview 8.4 Bio-Rad 8.4.1 Company Basic Information, Manufacturing Base and Competitors 8.4.2 Product Type, Application and Specification 8.4.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.4.4 Business Overview 8.5 CellaVision AB 8.5.1 Company Basic Information, Manufacturing Base and Competitors 8.5.2 Product Type, Application and Specification 8.5.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.5.4 Business Overview 8.6 Thermo Fisher Scientific, Inc. 8.6.1 Company Basic Information, Manufacturing Base and Competitors 8.6.2 Product Type, Application and Specification 8.6.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.6.4 Business Overview 8.7 EMD Millipore 8.7.1 Company Basic Information, Manufacturing Base and Competitors 8.7.2 Product Type, Application and Specification 8.7.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.7.4 Business Overview 8.8 Horiba Ltd. 8.8.1 Company Basic Information, Manufacturing Base and Competitors 8.8.2 Product Type, Application and Specification 8.8.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.8.4 Business Overview 8.9 Mindray Medical International Limited 8.9.1 Company Basic Information, Manufacturing Base and Competitors 8.9.2 Product Type, Application and Specification 8.9.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.9.4 Business Overview 8.10 Ortho-Clinical Diagnostics, Inc. 8.10.1 Company Basic Information, Manufacturing Base and Competitors 8.10.2 Product Type, Application and Specification 8.10.3 Sales, Revenue, Price and Gross Margin (2011-2016) 8.10.4 Business Overview 8.11 QIAGEN N.V. 8.12 Siemens Healthcare 8.13 Grifols, S.A 8.14 Dako Denmark A/S 8.15 eBioscience, Inc. 8.16 Sysmex Corporation 8.17 F. Hoffmann-La Roche Ltd. Get It Now @ https://www.wiseguyreports.com/checkout?currency=one_user-USD&report_id=729623
News Article | November 10, 2016
Generation of Ptpn11E76K-neo/+mice have previously been reported5. A neo cassette with a stop codon flanked by loxP sites was inserted in the second intron of the Ptpn11 allele followed by the mutation GAA (E) to AAA (K) at the amino acid 76 encoding position in the third exon. The mice were backcrossed to C57BL/6 mice for more than 10 generations. Ptpn11D61G/+ mice4 were originally imported from Beth Israel Deaconess Medical Center. Nestin-Cre+30, Mx1-Cre+31, Vav1-Cre+32, Prx1-Cre+33, Lepr-Cre+34, Osx1-Cre+35, Oc-Cre+36, and VE-Cadherin-Cre+-ERT2 (ref. 37) transgenic mice used in this study were purchased from the Jackson Laboratory or obtained from the investigators who originally developed the mouse lines. Mice of the same age, sex, and genotype were mixed and then randomly grouped for subsequent analyses (investigators were not blinded during allocation, during experiments and outcome assessment). All mice were kept under specific-pathogen-free conditions in the Animal Resources Center at Case Western Reserve University and subsequently Emory University Division of Animal Resources. All animal procedures complied with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Ptpn11E76K/+Mx1-Cre+ mice and Ptpn11+/+Mx1-Cre+ littermates (8 weeks old) were administered with i.p. injection of 3 doses of pI–pC (1.0 μg per g body weight) every other day over 5 days. Ptpn11E76K/+VE-Cadherin-Cre+-ERT2 mice and Ptpn11+/+VE-Cadherin-Cre+-ERT2 littermates (4–6 weeks old) were administered with i.p. injection of 3 doses of tamoxifen (9.0 mg per 40 g body weight) every other day over 5 days. Mice were analysed at the indicated time points after pI–pC or tamoxifen administration. Acute leukaemia progression in pI–pC administered Ptpn11E76K/+Mx1-Cre+ and Ptpn11E76K/+Vav1-Cre+ mice was determined as we previously described5. No statistical methods were used to predetermine sample size. De-identified BM biopsies from PTPN11-mutation-positive Noonan syndrome patients with JMML or non-syndromic PTPN11 mutation-positive patients with JMML were obtained from the University of California, San Francisco Tissue Cancer Cell Bank and Children’s Healthcare of Atlanta, Emory University. Informed consent was obtained from all subjects. The experiments involving human subjects were reviewed and approved (Exemption IV) by the Institutional Review Board of Emory University. BM cells (2 × 106) collected from indicated donor mice were transplanted into lethally irradiated (1,100 cGy) recipient mice with the indicated genotypes through tail vein injection. Recipients were monitored for MPN development for 6–8 months. To determine the abundance of the neo cassette in the targeted Ptpn11 allele, genomic DNA of haematopoietic cells, MSPCs, or other indicated cells was extracted with a ZR-Duet DNA/RNA MiniPrep extraction kit (Zymo Research). The abundance of the neo cassette was then quantified by qPCR using the Applied Biosystems 7500 Fast Real-Time PCR System. The PCR primers used were: 5′-TGGGAAGACAATAGCAGGCA-3′ and 5′-CCCACTCACCTTGTCATGTA-3′. The pool size, cell cycle status, apoptosis, and cell signalling activities of HSCs were analysed by multiparameter FACS analyses, as previously described38. In brief, for the HSC-pool-size analysis, fresh BM cells were stained with the following antibodies (eBiosciences, San Diego, unless otherwise noted): lineage antibodies (B220 (RA3-6B2), CD3 (145-2C11), Gr-1 (RB6-8C5), Mac-1 (M1/70), and Ter-119 (TER-119)), anti-Sca-1 (D7, BD Biosciences), anti-c-Kit (2B8), anti-CD150 (TC15-12F12.2, BD Biosciences), anti-CD48 (HM48-1), and anti-Flk2 (A2F10.1). Lin−Sca-1+c-Kit+CD150+CD48−Flk2− cells were quantified as HSCs. For the cell cycle analysis, freshly collected BM cells were stained for HSCs as above. Cells were then fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), stained with Ki-67 antibody, and further incubated with Hoechest 33342 (20 μg ml−1). For the apoptosis analysis, BM cells were stained for HSCs, and then incubated with Annexin V and 7-amino-actinomycin D (BD Biosciences). For cell signalling analyses, BM cells were stained for HSCs, fixed and permeabilized using a Cytofix/Cytoperm kit, and then stained with anti-phospho-Erk (mouse IgG) (E-4, Santa Cruz Biotechnology), anti-phospho-Akt (rabbit IgG) (C31E5E, Cell Signaling), or anti-phospho-NF-κB (rabbit IgG) (93H1, Cell Signaling) antibodies, washed and further incubated with AlexaFluor488-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG) (Life technologies). Phosphorylation levels of these signalling proteins were determined by mean fluorescence intensities (MFI) of gated cells. Data were collected on BD LSR II Flow Cytometer (BD Biosciences) and analysed with FlowJo (Treestar). HSCs (Lin−Sca-1+c-Kit+CD150+CD48−Flk2−) sorted from wild-type C57BL/6 mice were cultured in StemSpan medium supplemented with SCF (50 ng ml−1), Flt3 ligand (50 ng ml−1), TPO (50 ng ml−1), IL-3 (20 ng ml−1), and IL-6 (20 ng ml−1) in the presence of IL-1β (10 ng ml−1), CCL3 (20 ng ml−1), CCL4 (20 ng ml−1), or CCL12 (20 ng ml−1). Six days later, cells were collected and analysed for Mac-1+ myeloid cells, F4/80+ macrophages, and CD115+ monocytes. Mouse MSPCs were enriched following a standard protocol39. In brief, BM was collected from long bones. The bones were then crushed and digested with collagenase type II (2.5 mg ml−1) (Worthington Biochemical Corporation). BM cells and digested bone fragments were combined and cultured in DMEM supplemented with 15% fetal bovine serum (FBS). For human MSPC derivation, only BM cells were used. Suspension haematopoietic cells were removed after 24 h. Medium was replenished every 72 h. Colonies of MSPCs appeared 6–8 days after initial plating. To further purify MSPCs, cells were collected and stained with biotin-conjugated CD45 antibody and anti-biotin microbeads. CD45+ haematopoietic cells were depleted using MACS separation columns (Miltenyi Biotec Inc.). The purity of MSPCs (>95%) was further confirmed according to the (CD45−CD140α+Sca-1+) phenotypes39 by multiparameter FACS analyses. For the CFU-F assay, 2 × 106 unfractionated BM cells were plated and cultured for 10–14 days as described above. Cells were stained with 0.5% crystal violet (Sigma-Aldrich) in 10% methanol for 20 min. Colonies formed by more than 50 fibroblast-like cells were counted under a light microscope. For the CFU-GM assay, freshly collected BM cells (2 × 104 cells ml−1) were seeded in 0.9% methylcellulose IMDM medium containing 30% FBS, glutamine (10−4 M), β-mercaptoethanol (3.3 × 10−5 M), and IL-3 (1 ng ml−1) or GM-CSF (1 ng ml−1). After 7 days of culture at 37 °C in a humidified 5% CO incubator, colonies (primarily CFU-GM) formed by more than 50 haematopoietic cells were counted under an inverted microscope. MSPCs (CD45−Ter-119-CD31-CD140α+Sca-1+)39 were freshly isolated from the BM of Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice. RNA was extracted using the RNeasy Midi kit (Qiagen). Total RNA samples were enriched for polyadenylated transcripts using the Oligotex mRNA Mini kit (Qiagen), and strand-specific RNA-seq libraries were generated using PrepX RNA library preparation kits (IntegenX), following the manufacturer’s protocol. After cleanup with AMPure XP beads (Beckman Coulter) and amplification with Phusion High-Fidelity polymerase (New England BioLabs), RNA libraries were sequenced on a HiSeq 4000 instrument to a depth of at least 20 million reads. The correlation coefficient between the two groups is 0.954, which verifies that the method is accurate (Extended Data Fig. 6b). Before differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the DEGSeq R package (1.12.0). The P values were adjusted using the Benjamini–Hochberg method. Corrected P value of 0.005 and log (fold change) of 1 were set as the threshold for significantly different expression. Femurs were dissected from Ptpn11E76K/+Mx1-Cre+ mice and Ptpn11+/+Mx1-Cre+ littermates 12 weeks after pI–pC administration. BM plasma was collected by flushing one femur with 1.0 ml of phosphate buffered saline (PBS). MSPCs derived from pI–pC-administered Ptpn11E76K/+Mx1-Cre+ and Ptpn11+/+Mx1-Cre+ mice were cultured (4 × 106 cells in 2.0 ml medium) in serum-free DMEM for 48 h. The culture medium was then collected. BM plasma or MSPC culture medium were analysed with Mouse Cytokine Antibody Array blots (R&D Systems) following the instructions provided by the manufacturer. BM plasma collected from one femur and one tibia in 500 μl PBS. Culture medium was collected from mouse MSPCs (4 × 106 cells per 2.0 ml) at second or third passages cultured in serum-free DMEM for 48 h. These samples were assayed for levels of IL-1β and CCL3 using enzyme-linked immunosorbent assay (ELISA) kits (IL-1β: eBioscience; CCL3: R&D Systems) following the instructions provided by the manufacturers. To determine multiple cytokines/chemokines produced by human MSPCs, MSPCs (2 × 104 cells ml−1) were cultured in serum-free StemSpan medium for 72–96 h. To determine multiple protein factors produced by cells from patients with JMML, JMML cells (2 × 105 cells ml−1) were cultured in StemSpan medium supplemented with human SCF (50 ng ml−1), human Flt3 ligand (50 ng ml−1), and human TPO (50 ng ml−1) for 72 h. The culture medium was then collected and cytokine/chemokine levels were determined by the BD Cytometric Bead Array Flex Sets (BD Biosciences) following the manufacturer’s instructions. Human CXCL12 levels in MSPC culture medium were measured using a Human CXCL12/SDF-1 alpha Quantikine ELISA Kit (R&D systems). Frozen tissue sections prepared from 4% paraformaldehyde-fixed and decalcified bones were thawed at room temperature and then rehydrated with PBS. The slides were stained with the following antibodies (eBiosciences, San Diego, unless otherwise noted) following standard procedures: anti-Osteopontin (Abcam), anti-Nestin (MAB353, Millipore), anti-Gr-1 (RB6-8C5), anti-Mac-1 (M1/70), anti-B220 (RA3-6B2), anti-Ter-119 (TER-119), anti-CD3 (145-2C11, BD Biosciences), anti-CD115 (AFS98), anti-CD150 (TC15-12F12.2, BD Biosciences), anti-CD31 (MEC13.3, Biolegend), anti-CD48 (HM48-1), and anti-CD41 (eBioMWReg30) antibodies. Images were acquired using Olympus Confocal Laser Scanning Biological Microscope FV1000 equipped with four lasers ranging from 405 to 635 nm. Images were processed with ImageJ software. Ptpn11E76K/+Osx1-Cre+ mice (6–7 month old) and Ptpn11E76K/+Mx1-Cre+ mice (4 weeks after pI–pC administration) were treated daily via subcutaneous injection with the CCR1 antagonist BX471 ((2R)-1-((2-((aminocarbonyl)amino)-4-chlorophenoxy)acetyl)-4-((4-fluorophenyl)methyl)-2-methylpiperazine) purchased from Tocris Bioscience (50 mg kg−1 of body weight). These animals also received the CCR5 antagonist Maraviroc (4,4-difluoro-N-((S)-3-(3-(3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo(3.2.1)octan-8-yl)-1-phenylpropyl)cyclohexanecarboxamide) obtained from Selleck Chemicals (0.3 mg ml−1 in the drinking water). Control Ptpn11E76K/+Osx1-Cre+ mice and Ptpn11E76K/+Mx1-Cre+ mice were given vehicle (70% ethanol and 0.5% DMSO for subcutaneous injections, and 1% DMSO in drinking water). Mice were treated for 23 days and then killed for subsequent analyses. Data are presented as mean ± s.d. of all mice analysed in multiple experiments (that is, biological replicates). Statistical significance was determined using unpaired two-tailed Student’s t test. For HSC imaging analyses, two-tier tests were used to first combine technical replicates and then evaluate biological replicates. To determine statistical significance in the incidences of MPN development and malignant progression, Fisher’s exact tests were performed. *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant in Extended Data Figs 2, 5.
News Article | March 30, 2016
Filipin III was from Sigma. Amplex Red cholesterol assay kit was from Invitrogen. IL-2 was from Promega. For the flow cytometric analysis, anti-mCD4 (RM4-5), anti-mCD8 (53-6.7), anti-mCD3ε (145-2C11), anti-IFNγ (XMG1.2), anti-TNFα (MP6-XT22), anti-granzyme B (NGZB), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-PD-1 (J43), anti-CTLA-4 (UC10-4B9), anti-Ki-67 (16A8), anti-FoxP3 (FJK-16 s), anti-Gr1 (RB6-8C5), anti-CD11b (M1/70) and anti-CD45 (30-F11) were purchased from eBioscience. For western blots, anti-pCD3ζ, anti-CD3ζ, anti-pZAP70, anti-ZAP70, anti-pLAT, anti-LAT, anti-pERK1/2 and anti-ERK1/2 were from Cell Signaling Technology. Avasimibe was from Selleck. MβCD-cholesterol and MβCD were from Sigma. Lovastatin was from Sigma. U18666A was from Merck. K604 was chemically synthesized in F.-J. Nan’s laboratory. CP113,818 was a research gift from P. Fabre. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was from Promega. B16F10, Lewis lung carcinoma and EL-4 cell lines were originally obtained from the American Type Culture Collection, and proved mycoplasma-free. Listeria monocytogenes was provided by Q. Leng. C57BL/6 mice were purchased from SLAC. OT-I TCR transgenic mice were from the Jackson Laboratory. CD4cre transgenic mice was described previously31. InGeneious Labs produced homozygous Acat1flox/flox mouse. To produce this mouse, the Acat1 loxP construct was made by inserting two loxP sites covering Acat1 exon 14, which includes His460 known to be essential for the enzymatic activity32. The construct was injected into embryonic stem cells. The correctly targeted clones as determined by Southern blot and diagnostic PCR were injected into C57BL/6 blastocysts. To remove the Neo marker, the mice were further backcrossed to the C57BL/6 Frt mice. Through mouse crossing, the wild-type Acat1 allele (Acat1+/+), heterozygous Acat1 loxP allele (Acat1flox/+) and homozygous Acat1 loxP allele (Acat1flox/flox) were obtained and confirmed by using diagnostic PCR. Acat1flox/flox mice were crossed with CD4cre transgenic mice to get Acat1CKO mice with ACAT1 deficiency in T cells. Acat1CKO mice were further crossed with OT-I TCR transgenic mice to get Acat1CKO OT-I mice. Animal experiments using Acat1CKO mice were controlled by their littermates with normal ACAT1 expression (Acat1flox/flox). Animal experiments using Acat1CKO OT-I mice were controlled by their littermate with normal ACAT1 and OT-I TCR expression (Acat1flox/flox OT-I). Acat2−/− mice were purchased from Jackson Laboratory. All mice were maintained in pathogen-free facilities at the Institute of Biochemistry and Cell Biology. All animal experiments used mice with matched age and sex. Animals were randomly allocated to experimental groups. The animal experiments performed with a blinded manner were described below. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The maximal tumour measurements/volumes are in accordance with the IACUC. All human studies have been approved by the Research Ethical Committee from ChangZheng Hospital, Shanghai, China. Informed consent was obtained from all study subjects. Total RNA was extracted with Trizol (Life technology) from the indicated cells and subjected to quantitative reverse transcription PCR (qRT–PCR) using gene specific primers (5′–3′): Acat1 (forward, GAAACCGGCTGTCAAAATCTGG; reverse, TGTGACCATTTCTGTATGTGTCC); Acat2 (forward, ACAAGACAGACCTCTTCCCTC; reverse, ATGGTTCGGAAATGTTCACC); Nceh (forward, TTGAATACAGGCTAGTCCCACA; reverse, CAACGTAGGTAAACTGTTGTCCC); Srebp1 (forward, GCAGCCACCATCTAGCCTG; reverse, CAGCAGTGAGTCTGCCTTGAT); Srebp2 (forward, GCAGCAACGGGACCATTCT; reverse, CCCCATGACTAAGTCCTTCAACT); Acaca (forward, ATGGGCGGAATGGTCTCTTTC; reverse, TGGGGACCTTGTCTTCATCAT); Fasn (forward, GGAGGTGGTGATAGCCGGTAT; reverse, TGGGTAATCCATAGAGCCCAG); Hmgcs (forward, AACTGGTGCAGAAATCTCTAGC; reverse, GGTTGAATAGCTCAGAACTAGCC); Hmgcr (forward, AGCTTGCCCGAATTGTATGTG; reverse, TCTGTTGTGAACCATGTGACTTC); Sqle (forward, ATAAGAAATGCGGGGATGTCAC; reverse, ATATCCGAGAAGGCAGCGAAC); Ldlr (forward, TGACTCAGACGAACAAGGCTG, reverse, ATCTAGGCAATCTCGGTCTCC); Idol (forward, TGCAGGCGTCTAGGGATCAT; reverse, GTTTAAGGCGGTAAGGTGCCA); Abca1 (forward, AAAACCGCAGACATCCTTCAG; reverse, CATACCGAAACTCGTTCACCC); Abcg1 (forward, CTTTCCTACTCTGTACCCGAGG; reverse, CGGGGCATTCCATTGATAAGG); Ifng (forward, ATGAACGCTACACACTGCATC; reverse, CCATCCTTTTGCCAGTTCCTC). Three methods were used to measure the cholesterol level of T cells. Filipin III was dissolved in ethanol to reach the final concentration of 5 mg ml−1. Cells were fixed with 4% paraformaldehyde (PFA) and stained with 50 μg ml−1 filipin III for 30 min at 4 °C. Images were collected using a Leica SP8 confocal microscope and analysed using a Leica LAS AF software. The total cellular cholesterol level was quantified using the Amplex Red cholesterol assay kit (Invitrogen). To quantify the intracellular cholesterol, CD8+ T cells were fixed with 0.1% glutaraldehyde and then treated with 2 U ml−1 cholesterol oxidase for 15 min to oxidize the plasma membrane cholesterol. The intracellular cholesterol was then extracted with methanol/chloroform (vol/vol, 1: 2), and quantified using the Amplex Red cholesterol assay kit. The value of the plasma membrane cholesterol was obtained by subtracting the intracellular cholesterol from the total cellular cholesterol. Plasma membrane cholesterol level was measured as previously described33. The plasma membrane of CD8+ T cells was biotinylated by 1 mg ml−1 sulfo-NHS-S-biotin, and then the cells were lysed by passing 13 times through a ball-bearing homogenizer. Plasma membrane was isolated from the supernatant of homogenate by streptavidin magnetic beads. Lipids were extracted with hexane/isopropanol (vol/vol, 3: 2), and then were used for measurement of unesterified cholesterol with Amplex Red Cholesterol Assay Kit and choline-containing phospholipids with EnzyChrom Phospholipid Assay Kit. The relative plasma membrane cholesterol level was normalized to the total phospholipids. To deplete cholesterol from the plasma membrane, CD8+ T cells were treated with 0.1–1 mM MβCD for 5 min at 37 °C, and then washed three times with PBS. To add cholesterol to the plasma membrane, CD8+ T cells were incubated with the culture medium supplied with 1–20 μg ml−1 MβCD-coated cholesterol at 37 °C for 15 min. The cells were then washed three times with PBS. Peripheral T cells were isolated from mouse spleen and draining lymph nodes by a CD8+ or CD4+ T-cell negative selection kit (Stem cell). To analyse the tumour-infiltrating T cells, tumours were first digested by collagenase IV (sigma), and tumour-infiltrating leukocytes were isolated by 40–70% Percoll (GE) gradient centrifugation. To measure the effector function of CD8+ T cells, the isolated cells were first stimulated with 1 μM ionomycin and 50 ng ml−1 phorbol 12-myristate 13-acetate (PMA) for 4 h in the presence of 5 μg ml−1 BFA, and then stained with PERCP-conjugated anti-CD8a. Next, cells were fixed with 4% PFA and stained with FITC-conjugated anti-granzyme B, allophycocyanin (APC)-conjugated anti-IFNγ and phycoerythrin (PE)-conjugated anti-TNFα. In general, to gate the cytokine or granule-producing cells, T cells without stimulation or stained with isotype control antibody were used as negative controls. This gating strategy is applicable for most of the flow cytometric analyses. To detect the MDSC cells in the tumour, the Percoll-isolated leukocyte were stained with anti-CD45, anti-CD11b and anti-Ly6G (Gr1), the CD45+ population was gated, after which the MDSC population (CD11b+ Gr1+) in CD45+ were gated. A pan T-cell isolation kit (Miltenyi biotech) was used to deplete T cells from splenocytes isolated from C57BL/6 mice. The T-cell-depleted splenocytes were pulsed with antigenic peptides for 2 h and washed three times. SIINFEKL (OVA or N4), SAINFEKL (A2), SIITFEKL (T4), SIIGFEKL (G4) are four types of agonist antigens with strong to weak TCR affinities. RTYTYEKL (Catnb) is a self-antigen of OT-I TCR. SIIRFEKL (R4) supports the positive selection of OT-I T cells and thus mimics a self-antigen. The T-cell-depleted and antigen-pulsed splenocytes were co-incubated with Acat1CKO OT-I T cells or wild-type OT-I T cells for 24 h. Cytokine production of CD8+ T cells was measured by intracellular staining and flow cytometric analysis. To generate mature CTLs, splenocytes isolated from Acat1CKO OT-I mice or wild-type OT-I mice were stimulated with OVA (N4) for 3 days in the presence of 10 ng ml−1 IL-2. Cells were centrifuged and cultured in fresh medium containing 10 ng ml−1 IL-2 for 2 more days, after which most of the cells in the culture were CTLs. To measure CD8+ T-cell cytotoxicity, EL-4 cells were pulsed with 2 nM antigenic peptide (N4, A2, T4, G4, R4 or Catnb) for 30 min. After washing EL-4 cells and CTLs three times with PBS, we mixed CTLs and antigen-pulsed EL-4 cells (1 × 105) in the killing medium (phenol-free RPMI 1640, 2% FBS), at the ratios of 1:1, 2:1 and 5:1, respectively. After 4 h, the cytotoxic efficiency was measured by quantifying the release of endogenous lactate dehydrogenase (LDH) from EL-4 cells using a CytoTox 96 Non-Radioactive Cytotoxicity kit (Promega). Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg ml−1 phytohaemagglutinin (Sigma) for 2 days and then rested for 1 day. Cells were pretreated with vehicle (DMSO), CP113,818 or avasimibe for 12 h and then stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Intracellular staining and flow cytometry were used to measure cytokine productions of CD8+ T cells. Oxygen consumption rates and extracellular acidification rates were measured in nonbuffered DMEM (sigma) containing either 25 mM or 10 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate, under basal conditions and in response to 1 μM oligomycin (to block ATP synthesis), 1.5 μM FCCP (to uncouple ATP synthesis from the electron transport chain), 0.5 μM rotenone and antimycin A (to block complex I and III of the electron transport chain, respectively), and 200 μM etomoxir (to block mitochondrial fatty acid oxidation) on the XF-24 or XF-96 Extracellular Flux Analyzers (Seahorse Bioscience) according to the manufacturer’s recommendations. B16F10 cells (5 × 103) in 100 μl media containing avasimibe or DMSO were cultured for 24, 48 or 72 h. MTS reagent (20 μl) (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega) was added into each well. After a 2–3-h incubation, the absorbance at 490 nm was measured. The effect of avasimibe on cell viability was obtained by normalizing the absorbance of avasimibe-treated cells with that of the DMSO-treated cells. The viability value of DMSO-treated cells was set as 1. L. monocytogenes (2 × 104–7 × 104 colony-forming units (CFU)) expressing a truncated OVA protein were intravenously injected into Acat1CKO and littermate wild-type mice aged 8–10 weeks. On day 6, T cells isolated from spleens were stimulated with 50 ng ml−1 PMA and 1 μM ionomycin for 4 h in the presence of brefeldin A and then assessed by flow cytometry to detect IFNγ production. At the same time, the serum IFNγ level was assessed by ELISA. To detect the antigen-specific response of CD8+ T cells, the splenocytes were stimulated with 1 μM OVA peptide for 24 h. IFNγ production was analysed as mentioned above. To detect the L. monocytogens titre in the livers of infected mice, the livers were homogenized in 10 ml 0.2% (vol/vol) Nonidet P-40 in PBS, and the organ homogenates were diluted and plated on agar plates to determine the CFU of L. monocytogenes. Investigator was blinded to group allocation during the experiment and when assessing the outcome. B16F10 cells were washed three times with PBS, and filtered through a 40-μm strainer. In a skin melanoma model, B16F10 cells (2 × 105) were subcutaneously injected into the dorsal part of mice (aged 8–10 weeks). From day 10, tumour size was measured every 2 days, and animal survival rate was recorded every day. Tumour size was calculated as length × width. Mice with tumour size larger than 20 mm at the longest axis were euthanized for ethical consideration. To analyse effector function of tumour-infiltrating T cells, mice were euthanized on day 16. In the avasimibe therapy, melanoma-bearing mice with similar tumour size were randomly divided into two groups. From day 10, avasimibe was injected intraperitoneally to the mice at the dose of 15 mg kg−1 every 2 days. In a lung-metastatic melanoma model, B16F10 cells (2 × 105) were intravenously injected into mice (aged 8–10 weeks). Animal survival rate was recorded every day. To study tumour growth, mice were euthanized on day 20 and tumour numbers on lungs were counted. Lung-infiltrating T cells were isolated and analysed as mentioned above. In the lung-metastatic melanoma model, investigator was blinded to group allocation during the experiment and when assessing the outcome. B16F10-OVA cells (2 × 105) were injected subcutaneously into C57BL/6 mice at age 8–10 weeks. On day 16, the naive wild-type or Acat1CKO OT-I CD8+ T cells were isolated and labelled with live cell dye CFSE or CTDR (Cell Tracker Deep Red, Life Technologies), respectively. The labelled wild-type and CKO cells were mixed together at a 1:1 ratio, and 1 × 107 mixed cells per mouse were injected intravenously into the B16F10-OVA-bearing mice. After 12 h, blood, spleens, inguinal lymph nodes (draining) and mesenteric lymph nodes (non-draining) of the mice were collected. Single-cell suspensions from these tissues were stained with the anti-CD8a antibody, and the ratio of transferred cells in CD8+ populations was analysed using flow cytometry. The Lewis lung carcinoma cells were washed twice with PBS and filtered through a 40-μm strainer. After which, the Lewis lung carcinoma cells (2 × 106) were intravenously injected into wild-type or Acat1CKO mice at age 8–10 weeks. To detect the tumour multiplicity in the lung, the mice were euthanized at day 35 after tumour inoculation and tumour numbers in the lung were counted. In the avasimibe therapy, mice were randomly divided into two groups. From days 10 to 35 after tumour inoculation, avasimibe was delivered to the mice by intragastric administration at the dose of 15 mg kg−1 every 3 days. B16F10-OVA cells (2 × 105) were injected subcutaneously into C57BL/6 mice at age 8–10 weeks. On day 10, melanoma-bearing mice with similar tumour size were randomly divided into three groups (n = 9–10) and respectively received PBS, wild-type OT-I CTLs (1.5 × 106) or Acat1CKO OT-I CTLs (1.5 × 106) by intravenous injection. From day 13, the tumour size was measured every two days, and the animal survival rate was recorded every day. Tumour size was calculated as length × width. Mice with tumour size larger than 20 mm at the longest axis were euthanized for ethical consideration. B16F10 cells (2 × 105) were injected subcutaneously into C57BL/6 mice at age 8–12 weeks. On day 10, melanoma-bearing mice with similar tumour size were randomly divided into four groups (n = 8–10) and received PBS, avasimibe, anti-PD-1 antibody or both avasimibe and anti-PD-1 antibody, respectively. Avasimibe was delivered every 2 days at the dose of 15 mg kg−1 by intragastric administration. Anti-PD-1 antibody (RMP1-14, Bio X Cell, 200 μg per injection) was injected intraperitoneally every 3 days. The tumour size and survival were measured as mentioned above. Mice with tumour size larger than 20 mm at the longest axis were euthanized for ethical consideration. Super-resolution STORM imaging was performed on a custom modified Nikon N-STORM microscope equipped with a motorized inverted microscope ECLIPSE Ti-E, an Apochromat TIRF 100 × oil immersion lens with a numerical aperture of 1.49 (Nikon), an electron multiplying charge-coupled device (EMCCD) camera (iXon3 DU-897E, Andor Technology), a quad band filter composed of a quad line beam splitter (zt405/488/561/640rpc TIRF, Chroma Technology Corporation) and a quad line emission filter (brightline HC 446, 523, 600, 677, Semrock, Inc.). The TIRF angle was adjusted to oblique incidence excitation at the value of 3,950–4,000, allowing the capture of images at about 1 μm depth of samples. The focus was kept stable during acquisition using Nikon focus system. For the excitation of Alexa647, the 647 nm continuous wave visible fibre laser was used, and the 405 nm diode laser (CUBE 405-100C, Coherent Inc.) was used for switching back the fluorophores from dark to the fluorescent state. The integration time of the EMCCD camera was 90–95 frames per second. To image TCR distribution in the plasma membrane, naive CD8+ T cells or activated CD8+ T cells (stimulated with 10 μg ml−1 anti-CD3 for 10 min at 37 °C) were placed in Ibidi 35 mm μ-Dish and fixed with 4% PFA, followed by surface staining with 5 μg ml−1 anti-mCD3ε (145-2C11) for 4 h at 4 °C, then the cells were stained with 2 μg ml−1 Alexa 647-conjugated goat anti-hamster IgG (the secondary antibody) for 2 h at 4 °C after washing with PBS ten times. Before imaging, the buffer in the dish was replaced with the imaging buffer contained 100 mM β-mercaptoethanolamin (MEA) for a sufficient blinking of fluorophores. Super-resolution images were reconstructed from a series of 20,000–25,000 frames using the N-STORM analysis module of NIS Elements AR (Laboratory imaging s.r.o.). Molecule distribution and cluster position were analysed with MATLAB (MathWorks) based on Ripley’s K function. L(r) − r represents the efficiency of molecule clustering, and r value represents cluster radius. The r value at the maximum L(r) − r value represents the cluster size with the highest probability34. Planar lipid bilayers (PLBs) containing biotinylated lipids were prepared to bind biotin-conjugated stimulating antibody by streptavidin as previously described35, 36. Biotinylated liposomes were prepared by sonicating 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-cap-biotin (25:1 molar ratio, Avanti Polar Lipids) in PBS at a total lipid concentration of 5 mM. PLBs were formed in Lab-Tek chambers (NalgeNunc) in which the cover glasses were replaced with nanostrip-washed coverslips. Coverslips were incubated with 0.1 mM biotinylated liposomes in PBS for 20 min. After washing with 10 ml PBS, PLBs were incubated with 20 nM streptavidin for 20 min, and excessive streptavidin was removed by washing with 10 ml PBS. Streptavidin-containing PLBs were incubated with 20 nM bionylated anti-mCD3ε (145-2C11) (Biolegend). Excessive antibody was removed by washing with PBS. Next, PLBs were treated with 5% FBS in PBS for 30 min at 37 °C and washed thoroughly for TIRFM of T cells. Adhesion ligands necessary for immunological synapse formation were provided by treating the bilayer with serum. Freshly isolated mouse splenocytes were stained with Alexa568-anti-mTCRβ Fab and FITC-anti-mCD8 and washed twice. Anti-mTCRβ antibody was labelled with Alexa568-NHS ester (Molecular probes) and digested to get Fab fragments with Pierce Fab Micro Preparation Kit (Thermo). Cells were then placed on anti-mCD3ε-containing PLBs to crosslink TCR. Time-lapse TIRFM images were acquired on a heated stage with a 3-s interval time at 37 °C, 5% CO , using a Zeiss Axio Observer SD microscopy equipped with a TIRF port, Evolve 512 EMCCD camera and Zeiss Alpha Plan-Apochromat 100 × oil lens. The acquisition was controlled by ZEN system 2012 software. An OPSL laser 488 nm and a DPSS laser 561 nm were used. Field of 512 × 512 pixels was used to capture 6–8 CD8+ T cells per image. Results of synapse formation and TCR movements were the population averages of all CD8+ T cells from 2–3 individual images. The movements of TCR microclusters were splitted into directed, confined and random movement using the method described37. To sort the three movements, the MSD plot of each TCR microcluster was fitted with three functions as described37. The ones with good fit (square of correlation coefficients (R2) ≥ 0.33) were selected for further classification. For a certain TCR microcluster, the movement is defined as random if s.d. < 0.010. The distinction of directed and confined movement depends on which function fit better in the population of those s.d. ≥ 0.010. Images were analysed with Image Pro Plus software (Media Cybernetics), ImageJ (NIH) and MATLAB (MathWorks). In the granule polarization imaging, CTLs stained with Alexa568-anti-mTCRβ Fab were placed on anti-mCD3ε-containing PLBs for indicated time and fixed with 4% PFA. After the permabilization, cells were stained with Alexa488-anti-mCD107a (1D4B) antibody. Three-dimensional spinning-disc confocal microscopy was used to image the granules polarized at 0–2 μm distance from the synapse. The total granule volumes were quantified with Imaris software. The degranulation level was measured as previously described38. OT-I CTLs were mixed with OVA pulsed EL4 cells at 1:1 ratio. The mixed cells were then cultured in the medium supplemented with 1 μg ml−1 Alexa488-anti-CD107a antibody and 2 μM monensin for 1, 2 and 4 h. After which, cells were washed with PBS and further stained with PE–Cy7-anti-CD8a antibody. Flow cytometry was used for assessing the surface and internalized CD107a levels. MATLAB code used to perform STORM and TIRFM data analysis can be accessed by contacting W.L. (firstname.lastname@example.org). All sample sizes are large enough to ensure proper statistical analysis. Statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc.). Statistical significance was determined as indicated in the figure legends. P < 0.05 was considered significant; *P < 0.05; **P < 0.01; ***P < 0.001. All t-test analyses are two-tailed unpaired t-tests. The replicates in Figs 2, 3b, i, k–o, 4a, b, e–j, l, m and Extended Data Figs 1a, 3a–c, g–l, 4f, 5a–e, 6, 7g, 8, 9e, h, j and 10 were biological replicates. The replicates in Figs 1, 3c, d, p, Fig. 4o, p and Extended Data Figs 1b–i, 2, 3d–f, m, n, 4b–e, 5f, g, 7a, b, i–l and 9a–c were technical replicates. The centre values shown in all figures are average values.
News Article | September 7, 2016
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.).
News Article | February 22, 2017
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.
News Article | February 17, 2017
Research and Markets has announced the addition of the "Cell Analysis Global Market - Forecast to 2023" report to their offering. The cell analysis market is expected to grow at high single digit CAGR to reach $47,088 million by 2023. The major factor influencing the growth is enhanced precision of cell imaging and analysis systems which in turn reduce time and cost of drug discovery process. In addition, the factors like increasing incidence of cancer, increasing government investments, funds, and grants, availability of reagents and cell analysis instruments are driving the growth of the market. However, the major market restraints include high capital investments and a shortage of skilled labor for the high content screening procedure. The biggest opportunities for this market is the emerging APAC market, high content screening services provided by contract research organizations, automation in cancer research for its early diagnosis and reduction of cost in the cancer treatment. The cell analysis global market is a competitive and all the active players in this market are involved in innovating new and advanced products to maintain their market shares. The key players in the cell analysis global market include Agilent Technologies, Inc. (U.S.), Becton Dickinson and Company (U.S.), Bio-Rad Laboratories (U.S.), Danaher Corporation (U.S.), GE Healthcare (U.K.), Merck KGAA (Germany), Olympus Corporation (Japan), PerkinElmer, Inc. (U.S.), Promega Corporation (U.S.), Qiagen N.V. (Netherlands) and ThermoFisher Scientific, Inc. (U.S.). In order to offer the products with better software, most of the players in the cell analysis market are collaborating with companies and educational institutions. - 4titude (U.K.) - AB Sciex (U.S.) - Abbott Laboratories, Inc. (U.S.) - Abcam PLC (U.S.) - Abdos (India) - Abnova Corporation (Taiwan) - ACEA Bioscience, Inc (U.S.) - Active Motif (U.S.) - Adnagen (U.S.) - Advanced Cell Diagnostics (U.S.) - Agilent Technologies, Inc. (U.S.) - Alere (U.S.) - Analytik Jena AG (Germany) - Apocell (U.S.) - Applied Microarrays (U.S.) - Ausragen (U.S.) - Auxilab S.L (Spain) - Avantes BV (Netherlands) - Aven Inc (U.S.) - Aviva Bioscience (U.S.) - Becton Dickinson and Company (U.S.) - BGI (China) - Bibby Scientific Limited (U.K.) - Bio Care Medical LLC (U.S.) - BioDot Inc. (U.S.) - Biofluidica (U.S.) - Biologics (China) - BioMerieux SA (Germany) - Bio-Rad Laboratories (U.S.) - Bioron (France) - Biosearch Technologies (U.S.) - BioView (Israel) - BMS microscopes (Netherlands) - Bruker (U.S.) - Canopus Bioscience (U.S.) - Capp ApS (Denmark) - Carl Zeiss AG (Germany) - Cell Signaling Technology, Inc. (U.S.) - Cell-Vu (U.S.) - Cherry Biotech (France) - Cisbio Bioassays (France) - Clearbridge BioMedics (Singapore) - Corning Inc (U.S.) - Creatv Microtech inc (U.S.) - Cyflogic (Finland) - Cynvenio Biosystems (U.S.) - Cytognos S.L. (Spain) - DaAn Gene (China) - Danaher Corporation (U.S.) - Danish Micro Engineering (Denmark) - Diagenode (Netherlands) - DiscoveRx (U.S.) - Domel (Slovenia) - Dragon Laboratory Instruments Ltd (China) - eBioscience, Inc., (U.S.) - Eppendorf (Germany) - Etaluma, Inc (U.S.) - Eurofins Scientific (Luxembourg) - EXIQON (Denmark) - FEI Company (U.S.) - Fluidgm Corporation (U.S.) - Fluxion Biosciences (U.S.) - GE Healthcare (U.K.) - Genedata AG (Switzerland) - Genemed Biotechnologies Inc (U.S.) - General Biologicals (Taiwan) - Gyros AB (Sweden) - Handyem (Canada) - Hausser Scientific (U.S.) - Herolab GmbH (Germany) - Hettich lab technology (Germany) - Hoffmann-La Roche (Switzerland) - HORIBA, Ltd. (Japan) - Illumina (U.S.) - Immunodiagnostics systems (France) - Jasco (U.S.) - Jena Biosciences (Germany) - JEOL, Ltd. (Japan) - Jasco Analytical Instruments (U.S.) - Kapa Biosystems (U.S.) - Keyence Corporation (U.S.) - Kyratec (Australia) - Labcon (U.S.) - Labnet International, Inc (U.S.) - Lubio Science (Switzerland) - Luminex Corporation (U.S.) - LW Scientific (U.S.) - Macrogen Inc (South Korea) - Medical Econet (Austria) - Meijo techno (U.K.) - Merck KGaA (Germany) - Mettler-Toledo, Inc. (U.S.) - Micro-shot Technology Ltd (China) - Miltenyil Biotec (Germany) - Nanostring Technologies (U.S.) - New England Biolabs (U.S.) - Nikon Corporation (Japan) - Olympus Corporation (Japan) - Optika SRL., (Italy) - Ortho Clinical Diagnostics (U.S.) - Ortoalresa (Spain) - Oxford Nanopore Technologies, Ltd. (U.K.) - Pacific Biosciences (U.S.) - Panagene (South Korea) - Park Systems (Korea) - PerkinElmer Inc (U.S.) - Pheonix (U.S.) - PicoQuant GmbH (Germany) - Promega Corporation (U.S.) - Qiagen N.V. (Netherlands) - Quest Diagnostics (U.S.) - R&D Systems (U.S.) - Rain Dance Technologies (U.S.) - Rheonix (U.S.) - Rigaku Corporation (Japan) - RR Mechatronics (Netherlands) - Sacace Biotechnologies (Italy) - Sanyo (Japan) - Scienion (Germany) - Scientific Specialities Inc (U.S.) - Seegene (South Korea) - Seimens Healthcare (Germany) - Separation Technology, Inc (U.S.) - Shimadzu Scientific Instruments (Japan) - Sigma Laborzentrifugen GmbH (Germany) - Sohn GmbH (Germany) - Sony Biotechnology (U.S.) - Sprenson Bioscience (U.S.) - Stemcell Technologies (Canada) - Sysmex (Japan) - Tecan (Switzerland) - The Western Electric & Scientific Works (India) - ThermoFisher Scientific Inc (U.S.) - Thorlabs (U.S.) - Toyo Gosei Co., Ltd (Japan) - TrimGen Genetic Diagnostics (U.S.) - Vision Scientific Co Ltd (Korea) - Visitron Systems Gmbh (Germany) - Waters Corporation (U.S.) - Yokogawa Electric Corporation (Japan) - Zymo Research (U.S.) For more information about this report visit http://www.researchandmarkets.com/research/ngm5k6/cell_analysis About Research and Markets Research and Markets is the world's leading source for international market research reports and market data. 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News Article | April 20, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were blinded to allocation of mice for assessment of histopathology and readouts of inflammation. E. coli strains were routinely cultured aerobically at 37 °C in lysogeny broth (LB) and on LB agar plates. B. abortus was cultured in tryptic soy broth or on tryptic soy agar (TSA) plates,. Chlamydia muridarum strain Nigg II was purchased from ATCC (Manassas, VA). Bacteria were cultured in HeLa 229 cells in DMEM supplemented with 10% FBS. Elementary bodies (EBs) were purified by discontinuous density gradient centrifugations as described previously23 and stored at −80 °C. The HEK293 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS at 37 °C in a 5% CO atmosphere. HEK293 cells (ATCC CRL-1573) were obtained from ATCC and were grown in a 48-well tissue culture plates in DMEM containing 10% FBS until ~40% of confluency was reached. HEK293 cells were transfected with a total of 250 ng of plasmid DNA per well, consisting of 25 ng of the reporter construct pNF-κB-luc, 25 ng of the normalization vector pTK-LacZ, and 200 ng of the different combinations of mammalian expression vectors carrying the indicated gene (empty control vector, pCMV-HA-VceC5, pCMV-HA-TRAF2DN (this study), hNOD1-3×Flag, hNOD2-3×Flag, pCMV-HA-hRip2, hNOD1DN-3×Flag, hNOD2DN-3×Flag or pCMV-HA-Rip2DN24 and pCMV-myc-CDC42DN25. The dominant-negative form of TRAF2, lacking an amino-terminal RING finger domain26, was PCR amplified from cDNA prepared from HEK293 cells and cloned into the mammalian expression vector pCMV-HA (BD Biosciences Clontech). Forty-eight hours after transfection, cells were lysed either without any treatment, or stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen) and MDP (10 μg ml−1, InvivoGen). After five hours of treatment the cells were lysed and analysed for β-galactosidase and luciferase activity (Promega). FuGene HD (Roche) was used as a transfection reagent according to the manufacturer’s instructions. Cell lines were monitored for mycoplasma contamination. Bone-marrow-derived macrophages (BMDMs) were differentiated from bone marrow precursors from femur and tibiae of C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbor, ME), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) as described previously27. For BMDM experiments, 24-well microtitre plates were seeded with macrophages at a concentration of 5 × 105 cells per well in 0.5 ml of RPMI media (Invitrogen, Grand Island, NY) supplemented with 10% FBS and 10 mM l-glutamine (complete RPMI) and incubated for 48 h at 37 °C in 5% CO . BMDMs were stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen), MDP (10 μg ml−1, InvivoGen), thapsigargin (1 μM and 10 μM, Sigma-Aldrich), dithiothreitol (DTT) (1 mM, Sigma-Aldrich), and LPS (10 ng ml−1, InvivoGen) with or without pre-treatment (30 min) of the cells with IRE1α kinase inhibitor KIRA6 (1 μM, Calbiochem), IRE1α endonuclease inhibitor STF-083010 (50 μM, Sigma-Aldrich), PERK inhibitor GSK2656157 (500 nM, Calbiochem) and tauroursodeoxycholate TUDCA (200 μM, Sigma-Aldrich) in the presence of 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA). After 24 h of stimulation, samples for ELISA and gene expression analysis were collected as described below. Preparation of the B. abortus wild-type strain 2308 and the ∆vceC mutant inoculum and BMDM infection was performed as previously described27. Approximately 5 × 107 bacteria in 0.5 ml of complete RPMI were added to each well containing 5 × 105 BMDMs. Microtitre plates were centrifuged at 210g for 5 min at room temperature in order to synchronize infection. Cells were incubated for 20 min at 37 °C in 5% CO , and free bacteria were removed by three washes with PBS, and the zero-time-point sample was taken as described below. After the PBS wash, complete RPMI plus 50 mg ml−1 gentamicin and 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA) was added to the cells, and incubated at 37 °C in 5% CO . For cytokine production assays, supernatant for each well was sampled at 24 h after infection. In order to determine bacterial survival, the medium was aspirated at the time point described above, and the BMDMs were lysed with 0.5 ml of 0.5% Tween 20, followed by rinsing each well with 0.5 ml of PBS. Viable bacteria were quantified by serial dilution in sterile PBS and plating on TSA. For gene expression assays, BMDMs were suspended in 0.5 ml of TRI-reagent (Molecular Research Center, Cincinnati) at the time points described above and kept at −80 °C until further use. At least three independent assays were performed with triplicate samples, and the standard error of the mean for each time point was calculated. All mouse experiments were approved by the Institutional Animal Care and Use Committees at the University of California, Davis, and were conducted in accordance with institutional guidelines. Sample sizes were determined based on experience with infection models and were calculated to use the minimum number of animals possible to generate reproducible results. C57BL/6 wild-type mice and Rip2−/− mice (The Jackson Laboratory), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) were injected intraperitoneally (i.p.) with 100 μl of 2.5 mg per kg body weight of thapsigargin (Sigma-Aldrich) at 0 and 24 h, and 4 h after the second injection the mice were euthanized and serum and tissues collected for gene expression analysis and detection of cytokines. Where indicated, mice were treated i.p. at 12 h before the first thapsigargin dose and 12 h before the second thapsigargin dose with the ER stress inhibitor TUDCA (250 mg per kg body weight). Female and male C57BL/6, Nod1+/−Nod2+/−, Nod1−/−Nod2−/− mice, and Rip2−/− mice aged 6–8 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Groups of five mice were inoculated i.p. with 0.2 ml of PBS containing 5 × 105 CFU of B. abortus 2308 or its isogenic mutant ∆vceC, as previously described28. At 3 days post-infection, mice were euthanized by CO asphyxiation and their serum and spleens were collected aseptically at necropsy. The spleens were homogenized in 2 ml of PBS, and serial dilutions of the homogenate were plated on TSA for enumeration of CFU. Spleen samples were also collected for gene expression analysis as described below. When necessary, mice were treated i.p. at day one and two post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor TUDCA (Sigma-Aldrich), or 10 mg per kg body weight of the IRE1α kinase inhibitor KIRA6 (Calbiochem) or vehicle control. For the placentitis mouse model, C57BL/6, Nod1+/−Nod2+/− and Nod1−/−Nod2−/− mice, aged 8–10 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Female Nod1+/−Nod2+/− mice were mated with male C57BL/6 mice (control mice) and female Nod1−/−Nod2−/− mice were mated with male Nod1−/−Nod2−/− mice (NOD1/NOD2-deficient), and pregnancy was confirmed by presence of a vaginal plug. At 5 days of gestation, groups of pregnant mice were mock infected or infected i.p. with 1 × 105 CFU of Brucella abortus 2308 or its isogenic mutant ∆vceC (day 0). At 3, 7 and 13 days after infection mice were euthanized by CO asphyxiation and the spleen and placenta of dams were collected aseptically at necropsy. At day 13 after infection (corresponding to day 18 of gestation), viability of pups was evaluated based on the presence of fetal movement and heartbeat, and fetal size and skin colour. Fetuses were scored as viable if they exhibited movement and a heartbeat, visible blood vessels, bright pink skin, and were of normal size for their gestational period. Fetuses were scored as non-viable if fetal movement, heartbeat, and visible blood vessels were absent, skin was pale or opaque, and their size for gestational period or compared to littermates was small, or they showed evidence of fetal reabsorption. Percentage of viability was calculated as [(number viable pups per litter/total number pups per litter) × 100]. At each time point, the placenta samples were collected for bacteriology, gene expression analysis and blinded histopathological analysis (Extended Data Fig. 6d). When indicated, mice were treated i.p. at days 5, 7 and 9 post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor tauroursodeoxycholate TUDCA (Sigma-Aldrich) or vehicle control. RNA was isolated from BMDMs and mouse tissues using Tri-reagent (Molecular Research Center) according to the instructions of the manufacturer. Reverse transcription was performed on 1 μg of DNase-treated RNA with Taqman reverse transcription reagent (Applied Biosystems). For each real-time reaction, 4 μl of cDNA was used combined with primer pairs for mouse Actb, Il6, Hspa5 and Chop. Real time transcription-PCR was performed using Sybr green and an ABI 7900 RT–PCR machine (Applied Biosystems). The fold change in mRNA levels was determined using the comparative threshold cycle (C ) method. Target gene transcription was normalized to the levels of Actb mRNA. Cytokine levels in mouse serum and supernatants of infected BMDMs were measured using either a multiplex cytokine/chemokine assay (Bio-Plex 23-plex mouse cytokine assay; Bio-Rad), or via an enzyme-linked immunosorbent assay (IL-6 ELISA; eBioscience), according to the manufacturer’s instructions. Cytotoxicity was determined by using a LDH release assay in supernatant of BMDMs treated as described above. LDH release assay was performed using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega), following manufacturer’s protocol. The percentage of LDH release was calculated as follows: Percentage of LDH release = 100 × (absorbance reading of treated well − absorbance reading of untreated control)/(absorbance reading of maximum LDH release control − absorbance reading untreated control). The kit-provided lysis buffer was used to achieve complete cell lysis and the supernatant from lysis-buffer-treated cells was used to determine maximum LDH release control. HeLa 229 cells (ATCC CCL-2.1) were cultured in 96-well tissue culture plates at a concentration of 4 × 104 cells per well in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% FBS. HeLa 229 cells were transfected with a total of 125 ng of pCMV-HA-Rip2DN or empty control vector per well. 24 h post-transfection HeLa 229 cells were treated with Dextran to enhance infection efficacy before they were infected with 1.7 × 105 Chlamydia bacteria per well. The plates were centrifuged at 2,000 r.p.m. for 60 min at 37 °C, then incubated for 30 min at 37 °C in 5% CO Supernatant was discarded and replaced with DMEM containing 1 μg ml−1 cyclohexine (Sigma Aldrich) and where indicated, 1 μM KIRA6, 10 μM thapsigargin or 10 μg ml−1 MDP, was added to cultures before incubation at 37 °C in 5% CO for 40 h. For gene expression assays, HeLa 229 cells were suspended in Tri-reagent (Molecular Research Center, Cincinnati) and RNA was isolated. Infection efficiency was confirmed in separate plates by staining Chlamydia-infected HeLa 229 cells with anti-Chlamydia MOMP antibody and counting bacteria under a fluorescent microscope. Four independent assays were performed and the standard error of the mean calculated. BMDMs stimulated where indicated with 10 μM thapsigargin for 24 h were lysed in lysis buffer (4% SDS, 100 mM Tris, 20% glycerol) and 10 μg of protein was analysed by western blot using antibodies raised against rabbit TRAF2 (C192, #4724, Cell Signaling), rabbit HSP90 (E289, #4875, Cell Signaling), mouse SGT1 (ab60728, Abcam) and rabbit α/β-tubulin (#2148, Cell Signaling). For tissue culture experiments, statistical differences were calculated using a paired Student’s t-test. To determine statistical significance in animal experiments, an unpaired Student’s t-test was used. To determine statistical significance of differences in total histopathology scores, a Mann–Whitney U-test was used. A two-tailed P value of <0.05 was considered to be significant.
News Article | November 16, 2016
The murine cancer cell lines for melanoma (B16F10, referred to as B16), breast cancer (4T1) and colon cancer (CT26) were obtained from ATCC. The colon cancer cell line MC38 was obtained from the NCI and the lung cancer (LLC-Brei) from Caliper Life Sciences. Cells were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS) and penicillin with streptomycin (complete RPMI media). The GMCSF-secreting B16 cell line (referred as to B16-GMCSF) has been reported and was used to increase the number of myeloid cells recruited to the tumour14. The cell lines have been mycoplasma tested. cDNA was prepared using 1 μg RNA with the qScript cDNA Synthesis Kit (Quanta Biosciences). Sybr green-based qPCR was performed using murine primers to Arg1, Ifng, Il10, Il12p40, Il1b, Il6, Ccl2, Gapdh, Nos2, Tgfb1, Tnfa, IL4ra, Indo, Ctla4, Pd-l1, CD86, CxCr2, Fizz1, Ymd1 (Qiagen QuantiTect Primer Assay). mRNA levels were normalized to Gapdh (ΔC = C gene of interest – C Gapdh) and reported as relative mRNA expression or fold change. Tumours were excised, snap-frozen in liquid nitrogen, and pulverized using a tissue grinder. Tumour protein lysates were prepared in MSD Tris Lysis Buffer (Meso Scale Discovery, Rockville, Maryland) containing 2× Halt Protease and Phosphatase Inhibitor Cocktail (Fisher Scientific). Total protein concentration was normalized to 4 mg ml−1 and cytokines were quantified using the MILLIPLEX map Mouse Cytokine/Chemokine Magnetic Bead 32 Plex Panel and MILLIPLEX map Mouse CD8+ T Cell Magnetic Bead Panel kits according to the manufacturer’s instructions (Millipore, Billerica, Massachusetts). Mice bearing CT26 tumours were treated with vehicle or IPI-549 (15 mg kg−1 day−1, PO) for 6 or 9 days. Tumours were isolated, and frozen until needed; tumours then thawed and RNA was extracted from all cells. RNA-seq was done at Expression Analysis (Q2 Solutions). Sequence reads were aligned to the mouse B38 reference genome using OmicSoft ArrayStudio and the UCSC gene model. log [FPKM] was calculated for each gene, and data were mean centred for display in heat maps. The analysis focused on a compilation of about 4,200 mouse genes related to cancer immunology and PI3K pathway signalling compiled from numerous sources including BioCarta pathways, GO gene ontologies, KEGG pathways, WikiPathways, and literature23. Female C57BL/6J and Balb/c mice (6–8 weeks old) were purchased from Jackson Laboratory. Pmel-1 TCR transgenic mice have been previously reported24and were provided by N. Restifo (National Cancer Institute, Bethesda, Maryland). All mice were maintained in micro-isolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. No statistical methods were used to predetermine sample size. Ten to fifteen mice per treatment strategy were used to allow 90% power, and a 5% significance level, and detect differences in tumour-free survival from 10% to 80%. Typically, tumours grew in 100% of control animals. An additional 5 mice per group were used for tissue harvest at day 7 and day 14 on treatment. Mice cage and treatment allowance were randomized at day 7 after tumour implants. On day 0 of the experiments, tumour cells were injected intradermally (i.d.) in the right flank. For the B16 model, 2.5 × 105 B16-WT or B16-GMCSF cells were injected into C57BL/6J mice. For 4T1 model and for the CT26 model, 5 × 105 cells were used subcutaneously in Balb/c mice. For studies in immune compromised mice, the CT26 study was done in the Balb/c NU/NU strain and the B16-GMCSF in C57Bl.6 Rag1−/− mice. Mice were obtained from Jackson Labs and Charles River Labs. Treatments were given as single agents or in combinations with the following regimen for each drug. The PI3Kγ inhibitor drug IPI-549 was dissolved at 5% 1-methyl-2-pyrrolidinone in polyethylene glycol 400 and administered by oral gavage once a day at 15 mg kg−1. Treatment was initiated on day 7 ending on day 21 post tumour implant. Control groups received vehicle (5% NMP, 95% PEG) without the active product. Anti-CTLA4 antibody (100 μg per mouse, clone 9H10, Bio X cell) and anti-PD-1 antibody (250 μg per mouse, clone RPM1-14, Bio X cell) were injected intraperitoneally (i.p.) on days 7, 10, 13 and 16 for the B16, B16-GMCSF and 4T1 models and on days 10, 13 and 16 for the CT26 model. Tumours were measured every second or third day with a calliper, and the volume (length × width × height) was calculated. Mice that had no visible and palpable tumours that could be measured on consecutive measurement days were considered complete regressions. Animals were euthanized for signs of distress or when the total tumour volume reached 2,500 mm3. For the re-challenge study: mice with complete responses in the anti-PD-1 treatment group and the anti-PD-1 and IPI-549 combination group were re-challenged with 2.5 × 105 CT26 WT tumour cells (on day 106 of the study since original tumour implant). Additional mice with complete responses from an additional IPI-549 and anti-PD-1 group were implanted with 1 × 105 4T1 tumour cells. Female BALB/c mice were depleted of CD8+ T cells by intraperitoneal (IP) injection of a CD8-depleting antibody beginning 3 days before tumour implantation and continuing every 3 days (Q3D) for the duration of the study. Control animals were injected IP with an isotype control antibody according to the same dosing schedule. CD8+ T cell depletion was verified by flow cytometry of splenic cells from a separate cohort of mice at the time of tumour implant. Anti-CD8 antibody (BioXCell, in vivo MAb Rat IgG2b anti-mouse CD8, clone 169.4) and isotype control antibody (BioXCell, in vivo MAb Rat IgG2b, κ isotype control, clone LTF-2) were used. CD8+ T-cell-depleted and isotype contro-treated mice were implanted with 2.5 × 105 CT26 WT cells subcutaneously to the dorsal right flank. Eight days after implant, tumour-bearing animals with average tumour volumes of 50 to 60 mm3 began treatment. Animals were dosed daily with vehicle or IPI-549 (15 mg kg−1, PO) and dosed every three days with 100 μg antibody, either isotope control or anti-CD8. Tumour measurements were taken every second or third day during the 13 days of dose administration. For CD11b+ cell depletion, murine Lewis Lung Carcinoma tumour brei (here referred to as LLC) was propagated into ten C57BL/6 Albino male mice. When tumours reached an average of 1,500 mm3, tissue was collected and made into a single-cell suspension. Tissue was dissociated in a glass dunce, filtered through a 100 μm filter and washed with cold buffer of PBS pH 7.2, 0.5% BSA and 2 mM EDTA. An aliquot of cells was separated, and placed on ice until the time of re-transplant. Cells were counted and adjusted to 2 × 107 total cells. To deplete the CD11b cells from the tumour homogenate, CD11b micro beads (MACS #130-049-601) were added to the sample and incubated on ice for 15 min. Cells were washed and re-suspended with buffer. A negative depletion was preformed following the Miltenyi autoMACS protocol. The positive selected cells and starting tumour inoculum were counted and cell numbers adjusted to the tumour inoculum used for historical studies. For each cell condition 30 C57BL/Albino male mice were inoculated. Treatment of IPI-549 or vehicle started when tumours for each cell condition reached an average of 200 mm3 regardless of what day post implant this occurred. Mouse tumour samples were minced with scissors before incubation with 1.67 U ml−1 Liberase (Roche) and 0.2 mg ml−1 DNase (Roche) in RPMI for 30 min at 37 °C. Tumour samples were homogenized by repeated pipetting and filtered through a 100-μm nylon filter (BD Biosciences) in RPMI supplemented with 7.5% FCS to generate single-cell suspensions. Cell suspensions were washed once with complete RPMI and purified on a Ficoll gradient to eliminate dead cells. Cells from mouse spleens were isolated by grinding spleens through 40-μm filters. After red blood cell (RBC) lysis (ACK Lysing Buffer, Lonza) when required, all samples were washed and re-suspended in FACS buffer (PBS/0.5% albumin) or RPMI depending on further use. Cells isolated from mouse tumours and spleens were pre-incubated (15 min, 4 °C) with anti-CD16/32 monoclonal antibody (Fc block, clone 2.4G, BD Biosciences) to block nonspecific binding and then stained (30 min, 4 °C) with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD45-AF 700 (clone 30-F11), anti-CD11b-APC-Cy7 (clone M1/70), anti-CD11b-PE-TR(M1/70.15), anti-Ly6G-APC (clone 1A8), anti-F4/80-PercP-Cy5.5 (clone BM8), anti-Ly6C-PE-Cy7 (clone AL-21), anti-MHC Class II-eFluor 450 (clone M5/114.15.2), anti-CD206-PE (clone 19.2), anti-CD8-Percp-Cy5.5 (clone 53-6.7), anti-CD8-PE Texas Red (clone 5H10), anti-CD4-PE-Cy7 (clone RM4-5), anti-CD4-Pacific Blue (clone RM4-5), anti-Foxp3-APC (clone FJK-16 s), anti-Foxp3-PE-Cy7 (clone FJK-16 s), anti-CD25-APC-Cy7 (clone PC61), anti-CD44-PE-Cy7 (clone IM7), anti-CD62L-PE (clone MEL-14), anti-Ki67 (clone B56), anti-CTLA4-APC (clone), anti-Granzyme B-PE-TR (clone GB11), antibodies, all purchased from BD Biosciences, eBioscience or invitrogen. For intracellular stain, cells were further permeabilized using a FoxP3 Fixation and Permeabilization Kit (eBioscience) and stained for Foxp3 (clone FJK-16 s, Alexa-Fluor-700-conjugated, eBioscience), Ki67 (clone SolA15, eFluor-450-conjugated, eBioscience) or CTLA4. The stained cells were acquired on a LSRII Flow Cytometer using BD FACSDiva software (BD Biosciences) and the data were processed using FlowJo software (Treestar). Dead cells and doublets were excluded on the basis of forward and side scatter and Fixable Viability Dye eFluor 506. Mouse tumour and spleen single-cell suspensions were generated as described in the previous section. Tumour cells were subsequently separated from debris over a Ficoll gradient (Sigma-Aldrich). B cells were depleted from splenocytes using CD19 microbeads and LD columns according to the manufacturer’s instructions (Miltenyi Biotec) to enrich the myeloid fractions. Cells were stained with anti-CD45.2-Alexa- Fluor-700, anti-CD11b-APC-Cy7, anti CD8-PE antibodies for flow sorting on a FACSAria II Cell Sorter (BD Biosciences). Dead cells were excluded using DAPI (Invitrogen). Purity of flow-sorted populations was above 90%. Spleens and lymph nodes from pmel-1 TCR transgenic mice were isolated and ground through 100-μm filters. After RBC lysis, CD8+ T cells were purified by positive selection using Miltenyi magnetic beads. The isolated cells were loaded with CellTrace Far Red DDAO-s.e. (Thermo Fisher Scientific) and injected into recipient animals via tail vein at indicated numbers. Activated CD8+ T cells were generated by culturing splenocytes with soluble anti-CD3 (1 μg ml−1, clone 145-2C11, eBioscience) and anti-CD28 (2 μg ml−1, clone 37.51, eBioscience) for 72 h. Recombinant mouse IL-2 (30 U ml−1, Chiron) was added for the final 24 h of culture. CD8+ T cells were subsequently positively selected with Miltenyi magnetic beads before injection via tail vein, as described above. The frequency and proliferation of Pmel cells were measured 2 weeks after tumour challenge and 7 days after adoptive transfer of 1 × 106 in vitro activated CD8+ Pmel T cells using Thy1.1 antibody and by assessing CellTrace Far Red DDAO-s.e. dilution by flow cytometry, respectively. Spleens from naive mice were isolated and ground through 40-μm filters to generate a single-cell suspension. After RBC lysis, CD8+ cells were purified using anti-CD8 (Ly-2) microbeads (Miltenyi Biotech) according to the manufacturer’s protocol and labelled with 1 mM CFSE (Invitrogen) in pre-warmed PBS for 10 min at 37 °C. The CFSE-labelled CD8+ T cells were then plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round bottom 96-well plates (25 × 103 cells per well) coated with 1 μg ml−1 anti- CD3 (clone 1454-2C11) and 5 μg ml−1 anti-CD28 (clone 37N) antibodies. Purified myeloid cells were added in indicated ratios and plates were incubated at 37 °C. After 48 h, cells were harvested and CFSE signal in the gated CD8+ T cells was measured by flow cytometry (LSRII Flow Cytometer, BD Biosciences). For the human MDSC suppression assay PBMCs were isolated using Lymphoprep from donor blood. T cells were isolated by CD3+ selection (Easysep, Stem Cell Technologies) and frozen in Sigma freezing media for later use. The remaining PBMCs without T cells were incubated with 20 ng ml−1 GMCSF and 20 ng ml−1 IL-6 for 6 days to differentiate the myeloid cells and were incubated with or without added IPI-549. After 6 days, the MDSC cells were isolated by CD33+ selection (Easysep, Stem Cell Technologies). These MDSC cells were then mixed with the autologous T cells (at a 1:4 ratio) that had been pre-stained with cell trace violet and activated with anti-CD3 and anti-CD28 beads (Dynal). The T cell proliferation is determined by Cell Trace Violet dye dilution measured by flow cytometry after 72 h. Blood was collected from CT26 tumour-bearing mice after 10 days of treatment with either vehicle or IPI-549 (15 mg kg−1). PBMCs were isolated using Lymphoprep density gradient media (Stem Cell Technologies, Vancouver, BC). IFNγ producing cells were quantified using the CTL Mouse Immunospot IFNγ Single Colour ELISPOT kit (CTL, Shaker Heights, Ohio) according to the manufacturer’s instructions. For in vitro re-stimulation, 1 × 105 PBMC were co-cultured with 1 × 105 irradiated (2,000 rad) CT26 colon carcinoma target cells in CTL test media (CTL) for 16 h. Irradiated (2,000 rad) 4T1 mammary carcinoma target cells were used as negative control targets to assess specificity. IFNγ spots were quantified using a CTL Immunospot S6 Micro Analyzer and Immunospot Professional Software (CTL). Bone marrow derived macrophages were prepared from C57Bl/6 mice femur and tibias. Red blood cells were lysed and then the remaining cells were plated in bone macrophage media (BMM) consisting of DMEM, 20% FBS plus penicillin/streptomycin and 50 ng ml−1 M-CSF and incubated for 6 days. Cells were polarized towards an M2 phenotype with the addition of 20 ng ml−1 IL4 and 50 ng ml−1 M-MCSF (both from R&D Systems) with or without added IPI-549. Cells were incubated for 48 h and then RNA was harvested from the cells (Qiagen RNeasy). qRT–PCR was performed using primers for mouse ARG1 (Mm00475988_m1, Life Technologies NY) and mouse β-actin (Mm00607939_s1 Life Technologies, New York). Biochemical and cell-based assays for the Class I and Class II PI3K isoforms were run as previously described19, 25. Whole blood from six healthy donors was pre-treated with a dose titration of IPI-549 and then stimulated for 2 min with 2.3 μg ml−1 CXCL12. Cells were lysed, fixed and stained. Human blood samples were collected after ICF approval. Response to stimulation was determined by measuring phosphorylation of AKT S473 in monocytes (CD14+) by flow cytometry and comparing the value to that of untreated controls. IC values for IPI-549 were calculated for each donor, measuring compound potency against PI3Kγ and in whole blood. Where indicated, data were analysed for statistical significance and reported as P values. Data were analysed with a Mann–Whitney test when comparing means of two independent groups and two-way ANOVA when comparing more than two groups. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Evaluation of survival patterns in tumour-bearing mice was performed using the Kaplan–Meier method, and results were ranked according to the Mantel–Cox log-rank test. P < 0.05was considered statistically significant. Survival was defined as mice with tumours <2,500 cm3. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments have been repeated to ensure reproducibility of the observations. Source Data for the main figures are provided in the online version of this paper.