Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Orai1 protein sequences aligned with CLustalW algorithm for the indicated organisms. Cysteines susceptible to be S-acylated are highlighted in yellow. (B) Schematic ORAI1 representation. Superimposed structures of the WT and H206A dOrai (PDB ID: 4HKR and 6BBF) conformations in ribbon representation highlighting cysteine residues at position 126, 143 and 195 (C) Fluorescence images of WT O1/S1 cells (left) and averaged mCh-STIM1 and ORAI1-GFP fluorescence of the different O1/S1 stable cell lines. (D) Averaged fura-2 responses (left) and peak amplitude (right) of O1/S1 cells bearing or not the indicated ORAI1 mutation(s). Data are mean±SEM of 44-82 cells from two independent experiments. One way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Fluorescence, Stable Transfection, Mutagenesis

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) ORAI1 immunoblot of HeLa cells treated with PEG-5k to label S-acylation sites after exposure to NEM to block free thiols and then to hydroxylamine (HA) to break acyl-thioester bonds. (B) Western blot and corresponding autoradiogram of HeLa cells labelled for 2 h with H-palmitic acid with or without HA and immunoprecipitated with anti-ORAI1. (C, D) Western blots and corresponding autoradiograms of HeLa (C) and RPE-1 (D) cells expressing the indicated GFP-tagged ORAI1 mutants labelled with H-palmitic acid and immunoprecipitated with anti-GFP. Blots are representative of 3 independent experiments. (E) Normalized mean fura-2 responses evoked by Ca 2+ readmission in HEK-TKO cells transiently transfected with the indicated ORAI1-GFP constructs and exposed to Tg. (F) Peak amplitude of the responses in E after background subtraction. Data are mean±SEM of 8 independent experiments. One way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Western Blot, Blocking Assay, Immunoprecipitation, Expressing, Transfection, Construct

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Normalized fura-2 responses evoked by Ca 2+ readmission to Tg-treated HEK-293 cells stably expressing mCh-STIM1 and ORAI1-GFP (O1/S1) bearing or not the C143A mutation. (B) Peak amplitude of the responses in A. Data are mean±SEM of 196 (WT) and 198 (C143A) cells from 5 independent experiments. (C) Representative I CRAC recordings of WT and C143A O1/S1 cells, measured every 5 seconds at −100 mV. I CRAC was activated by cell dialysis with 10 mM BAPTA and blocked by 10 µM Gd 3+ . (D) Current-voltage relationship of the peak current in the cells shown in C (mean±SEM). (E) Peak current densities (I max ) of WT and C143A O1/S1 cells after subtraction of basal or Gd 3+ -insensitive currents. (F) Time-course of current activation in cells without pre-activated currents. Left: Recordings were aligned to the first inflexion point and basal and maximal values set to 0 and 1, respectively. Right: Statistical evaluation of the activation time. Data are mean±SEM, number of cells is indicated on the graphs. Two-tailed unpaired Student’s t -test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Stable Transfection, Expressing, Mutagenesis, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Representative TIRF images of WT and C143 O1/S1 cells exposed to 10 µM CPA for 10 min to induce mCh-STIM1 and ORAI1-GFP clustering Bars = 5 µm. (B) Averaged size of individual ORAI1-GFP clusters (left) and extent of PM covered by clusters (right) after CPA treatment. Data are mean±SEM of 29 (WT) and 30 (C143A) cells from 3 independent experiments. (C) FRAP recordings from WT and C143 O1/S1 cells. Top: representative GFP images. Bottom: representative fluorescence decay and recovery (left), diffusion coefficients (middle), and fluorescence plateau values (right). Data are mean±SEM of 21 (WT) and 15 (C143A) cells from 3 independent experiments Bars = 5 µm. (D) Lipid partitioning of ORAI1 in giant vesicles from HEK-293 cells transiently transfected with WT or C143 ORAI1-GFP. Top: representative fluorescence images of vesicles from cells expressing WT or C143 ORAI1-GFP (green) stained with cholera toxin subunit B (red) as raft marker and DiD (white) as non-raft marker top bar = 3 µm; bottom bar = 2µm. Bottom: Manders co-localization index for the indicated staining and conditions. Data are mean±SEM of 11 (WT) and 10 (C143A) vesicles from 3 independent experiments. Two-tailed unpaired Student’s t -test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Fluorescence, Diffusion-based Assay, Transfection, Expressing, Staining, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: Time-course of CPA-induced changes in the number, size (μm ), and extent of PM covered by ORAI1-GFP (left) and mCh-STIM1 (right) clusters in WT and C1434A O1/S1 cells. Data are mean±SEM of 29 (WT) and 30 (C143A) cells from 3 independent experiments. Two-Way ANOVA fitting mixed model.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques:

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Western blot and matching autoradiogram of RPE-1 cells expressing ORAI1-GFP plus the indicated PAT isoform, labelled with H-palmitic acid and immunoprecipitated with anti-GFP. Representative of 3 independent experiments. (B) Functional effect of PAT3, 7, and 20 expression. Western blot of HeLa cells expressing Myc-tagged PAT isoforms (left), averaged SOCE responses (middle), and peak amplitude (right). Data are mean±SEM of 49-74 cells from 3 independent experiments. (C) Averaged SOCE responses of WT (left) or C143A (middle) S1/O1 cells expressing these PAT isoforms and their peak amplitude (right). Data are mean±SEM of 31-129 cells from 5 independent experiments. (D) Confocal images of HeLa cells expressing Myc-tagged PAT20, treated or not with Tg (600s). Graphs show co-localization coefficients of Myc immunoreactivity with ORAI1-GFP, indicated by arrows on images. Bar = 10 µm. One way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Western Blot, Expressing, Immunoprecipitation, Functional Assay

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) 3 H-palmitate incorporation in RPE-1 cells expressing ORAI1-GFP plus the indicated PAT isoforms as in , normalized for expression levels. Data are from 2 independent experiments. (B) PAT3, PAT7, and PAT20 expression levels (left), averaged SOCE responses (middle), and peak amplitude (right) in HEK-293T-S1/O1-WT cells transfected with the indicated siRNAs. Data are mean±SEM of 61-126 cells from 2 independent experiments. One way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) ORAI1, PAT3, PAT7, and PAT20 abundance in proteomes from different tissues (from http://www.humanproteomemap.org/ consulted on Dec. 16, 2020). (B) Sequences of genomic DNA used to generate the CRISPR ORAI1 Jurkat T cell lines (top) and FLAG immunoblot of Jurkat T cells expressing FLAG-tagged Cas9 (bottom). (C) Representative flow cytometry Fluo 8 responses evoked by the Tg/Ca 2+ protocol in the indicated cells (top) and their averaged response and peak amplitude (bottom). Data are mean±SD of 2 independent experiments. (D) Fluorescence intensity profiles of CRISPR ORAI1 cells reconstituted with WT and C143 ORAI1-GFP measured by flow cytometry (N = 4). (E) Fluorescence images of CRISPR ORAI1 cells reconstituted with WT and C143 ORAI1-GFP cells blotted against NFATC1 ab treated or not with Tg to induce nuclear translocation of NFATC1 Bar = 10 µm. (F) Averaged SOCE responses (left) and peak SOCE amplitude (right) measured with YC3.6 in indicated cells expressing PAT20 or the empty vector (PCDNA3) (CRISPR Control + vector, 34 cells; CRISPR Control +PAT20, 40 cells; CRISPR ORAI1 +vector, 10 cells; CRISPR ORAI1+PAT20, 12 cells). (G) IL-2 positive cells in same cells as F treated with Tg or CD3/CD28 beads plus Ionomycin 1µM +PMA 20nM for 2h. One way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: CRISPR, Western Blot, Expressing, Flow Cytometry, Fluorescence, Translocation Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Averaged fura-2 responses and their peak amplitude evoked by Tg in Jurkat T cells lines generated by CRISPR with control or ORAI1-targeted guiding sequences and stably re-expressing either WT or C143A ORAI1-GFP. (B) Representative Fura-2 recordings of the indicated cell lines exposed to CD3/CD28-coated beads in physiological saline (left). Graph bars show the peak values evoked by CD3/CD28 beads in individual cells during the recording period. The percentages of cells with one or more elevation exceeding a threshold of 150% above basal is indicated. Data are from 102 cells (WT) and 124 cells (C143A) from 3 independent experiments. (C) Representative Fura-2 recordings of indicated cells exposed to CD3/CD28 beads in Ca 2+ -free media and then to 1 mM and 2 mM Ca 2+ (left), and peak amplitude of these responses (right). Graph data are mean±SEM of 228 cells (WT) and 185 cells (C143A) from 3 independent experiments. (D) NFATC1 translocation evoked by Tg in the indicated cell lines. Data are mean±SEM of the nuclear to cytosol NFATC1-GFP intensity ratio of 58-161 cells from 4 independent experiments. (E) Time-course of NFATC1 translocation evoked by CD3 (OKT3 1µg/ml). Data are mean±SEM of 51-132 cells from 3 independent experiments. (F) IL-2 production evoked by CD3 (OKT3 1µg/ml). Data are mean±SEM of 3 independent experiments. One way ANOVA Dunnett’s multiple comparisons test (A and D) or two-tailed unpaired Student’s t -test.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Generated, CRISPR, Stable Transfection, Expressing, Translocation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Confocal images of ORAI1-deficient Jurkat T cells reconstituted with WT or mutant ORAI1-GFP, stained with SiR-Actin during initial contact with CD3/CD28-coated beads (visible by their autofluorescence in the GFP channel). Graphs show fluorescence intensities along IS-centred transcellular sections indicated by rectangles on GFP images. Dotted lines on SiR-Actin image indicate the zoomed regions. (B) Time-course of ORAI1-GFP (left) and Sir-Actin (right) accumulation at synapses forming in cells reconstituted with ORAI1-WT (5 cells) and ORAI1-C143A (3 cells). Two-ways ANOVA. (C) TIRF images of these cells stained with Sir-Actin and then with anti-TCR mAb after plating on activating coverslips coated with anti-CD3 mAb. Sketches show densities of TCR and ORAI1 in different concentric regions within the IS (Bar = 10 µm). (D) Averaged ORAI-GFP fluorescence and (E) numbers of TCR clusters within IS forming in these two Jurkat T cell lines. (F) Manders co-localisation index for TCR and ORAI1 WT or mutant. Data are mean±SEM of 28-61 cells from four independent experiments. Two-tailed unpaired Student’s t -test. (G) Scheme representing the effect of ORAI1 S-acylation on IS molecular composition and function. Addition of palmitate to ORAI1 channels by PAT20 targets the channel to lipid-ordered PM domains, promoting the formation of concentric ORAI1 and TCR clusters engaging MHC at the immune synapse. The resulting sustained local Ca 2+ elevations (in red) induce nuclear translocation of NFATC1 to trigger IL-2 production. (H) Scheme representing the observed phenotype in TIRFF, where C143A mutant cells would accumulate less ORAI1 at the IS, it would redistribute in the centre and would have les TCR dots.

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: Mutagenesis, Staining, Fluorescence, Two Tailed Test, Translocation Assay

Journal: bioRxiv

Article Title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

doi: 10.1101/2021.02.03.429577

Figure Lengend Snippet: (A) Fraction of CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-GFP forming actin rings upon plating onto activating coverslips (WT = 258 cells C143A = 235 cells). (B) Averaged IS area of indicated cells forming actin rings. (C) Percentage of ORAI1 (left) and TCR (middle) signal originating from different concentric regions within the IS depicted in the sketch (right). WT = 44 cells; C143A = 27 cells. (D) TCR-PE intensity profiles of these cell lines (N = 3). Data are mean±SEM of three experiments. Two-tailed unpaired Student’s t -test (C) or Fisher’s exact test (A).

Article Snippet: CRISPR Jurkat T cells were generated by stably expressing with lentiviral particles pLX-311-Cas9 construct (Addgene 96924) and transiently transfecting with Amaxa® Cell Line Nucleofector® Kit T (Ref: VCA-1002, Lonza) two sets of sgRNAs (Hs.Cas9.ORAI1.1.AA Ref: 224748421 / Hs.Cas9.ORAI1.1.AB Ref: 224748422, IDT).

Techniques: CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Table summarizing the kidney-derived cancer cell lines characterized in CTRP, their sensitivities to GPX4 inhibitors and paclitaxel by AUC values and the cancer subtype information. * uncertain subtype; n.a., sensitivity value not available. Cell lines marked in red were used for individual validation in . B. Scatterplot of AUC value distributions for paclitaxel in all solid tumor cancer cell lines (sCCL, blue) or cell lines from each specified tissue-of-origin, including kidney (orange). Larger AUC values indicate lower compound sensitivity, and vice versa. Abbreviations: CNS, central nervous system; UAT, upper aerodigestive tract; a_ganglia, autonomic ganglia. A Mann-Whitney-Wilcoxon test was performed between each tissue and sCCLs from other tissues. Statistical significance is adjusted for multi-test correction. *, p<0.05; **, p<0.01; ****, p< 0.0001. Line and error bars: mean and standard deviation (S.D.). C. Relative viability of 786-O, 769-P, and OS-RC2 cells expressing shNC or shGPX4. Cellular viability was measured at 7 days post-lentiviral shRNA infection and normalized to shNC cells. Student’s t-test was performed between each shRNA and the shNC. ****, p<0.0001. Each condition has four biological replicates and error bars represent ±S.D. D. Relative sensitivity score (CERES score) of GPX4 knockout by CRISPR in cancer cell lines from indicated tissue-of-origin including kidney (red) from the Cancer Dependency Map (DepMap) database. Tissue abbreviations are the same as in panel C. E. Relative sensitivity score (ATARiS score) of GPX4 knockdown by shRNA in cancer cell lines from indicated tissue-of-origin including kidney (red) from the DepMap database. Tissue abbreviations are the same as in panel C. F. Confocal images of 786-O, 769-P, and BFTC909 cells treated with ML210 and indicated concentrations of vehicle (DMSO) or Lip-1 for the indicated time periods. Nine fields per treatment condition were aligned together for effective visualization. Scale bars in the images represent 50μm. G. Immunoblot analysis of HIF-2α protein levels in CCLF_KIPA_0001_N (normal), CCLF_KIPA_0001_T (tumor), and CCLF_KIPA_0002_T (tumor) cells. β-Actin was used as a loading control. Representative plot of experiments repeated three times.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Derivative Assay, MANN-WHITNEY, Standard Deviation, Expressing, shRNA, Infection, Knock-Out, CRISPR, Knockdown, Western Blot, Control

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Expression of GPX4 mRNA levels in the indicated cancer types including ccRCC (red) from The Cancer Genome Atlas (TCGA) database. Processed data were retrieved from cBioPortal. Log2 RPKM is presented for each sample. The cancer types are ordered by the mean of GPX4 expression. Cancer type abbreviations: Breast, Breast Invasive Carcinoma; DLBCL, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; Head&Neck, Head and Neck Squamous Cell Carcinoma; Liver HCC, Liver Hepatocellular Carcinoma; Liver CCC, Cholangiocarcinoma; Ovarian, Ovarian Serous Cystadenocarcinoma; Uterine CS, Uterine Carcinosarcoma; Glioma, Brain Lower Grade Glioma; Colorectal, Colorectal Adenocarcinoma; ccRCC, Kidney Renal Clear Cell Carcinoma; Lung adeno, Lung Adenocarcinoma; Prostate, Prostate Adenocarcinoma; Cervical, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; Bladder, Bladder Urothelial Carcinoma; Esophagus, Esophageal Carcinoma; Pancreas, Pancreatic Adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; GBM, Glioblastoma Multiforme; Thyroid, Thyroid Carcinoma; Uterine, Uterine Corpus Endometrial Carcinoma; Melanoma, Skin Cutaneous Melanoma; Stomach, Stomach Adenocarcinoma; Testicular, Testicular Germ Cell Cancer; pRCC, Kidney Renal Papillary Cell Carcinoma; AML, Acute Myeloid Leukemia; chRCC, Kidney Chromophobe; Lung squ, Lung Squamous Cell Carcinoma. B. Expression of relative GPX4 mRNA levels in cancer cell lines from each indicated tissue of origin including kidney (red) characterized in CTRP. The cancer types are ordered by the mean of GPX4 expression. Abbreviations for tissue of origin: CNS, central nervous system; UAT, upper aerodigestive tract; a_ganglia, autonomic ganglia. C. Immunoblot analysis confirming the expression of Cas9-V5 protein in 786-O and 769-P cells transduced with constitutive Cas9 expression. β-Actin was used as a loading control. D. Venn Diagram summarizing the number of genes that are significantly enriched under each combination of screening conditions. E. Volcano plot highlighting (red) the top enriched CRISPR hits comparing 4-day or 6-day ML210 treated 786-O cells with the DMSO-treated condition. LFC, log2 fold change (ML210/DMSO). F. qRT-PCR analysis of relative AGPAT3 mRNA expression in 769-P cells expressing shNC or AGPAT3-targeting shRNAs. *, p<0.05; **, p<0.01. B2M was used as a loading control. G. Viability curves for shNC or AGPAT3-targeting shRNA-expressing 769-P cells under indicated concentrations of ML210 or RSL3. Viability under each condition is relative to that of the respective DMSO-treated condition. Representative plot of experiments repeated three times. Each data point has four biological replicates, and error bars represent ±S.D.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Expressing, Western Blot, Transduction, Control, CRISPR, Quantitative RT-PCR, shRNA

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Experimental scheme describing the genome-wide CRISPR resistance screening to identify mediators of ML210 sensitivity in 786-O cells. 786-O cells were transduced with Cas9 expression, infected with a genome-wide lentiviral sgRNA library, selected with puromycin, and expanded for treatment with DMSO or ML210 for 4, 6, or 8 days. Forty million cells from the DMSO-treated conditions (minimum representation number per sgRNA> 500) were used as control samples, whereas cells that survived ML210-treatment were grown in drug-free media for one day before harvest. Genomic DNA was extracted from cell pellets and sgRNA barcode abundances were analyzed by sequencing. Top genes with sgRNAs enriched in ML210-treated cells were further validated. B. Volcano plot highlighting (red) the top enriched CRISPR hits comparing 786-O cells treated with ML210 or DMSO for 8 days. LFC, log2 fold change (ML210/DMSO). C. Immunoblot analysis of ACSL4 protein levels in vector (EV) and ACSL4-targeting sgRNA-expressing 786-O-Cas9 and 769-P-Cas9 cells. β-Actin was used as a loading control. D. Viability curves for WT (Vector) and ACSL4-targeting sgRNA-expressing 786-O or 769-P cells under indicated concentrations of ML210 or RSL3. E. Immunoblot analysis of KEAP1 and NRF2 (NFE2L2) protein levels in WT (Vector) or KEAP1-targeting sgRNA-expressing 786-O-Cas9 and 769-P-Cas9 cells. β-Actin was used as a loading control. F. Viability curves for WT (Vector) and KEAP1-targeting sgRNA-expressing 786-O-Cas9 or 769-P-Cas9 cells under indicated concentrations of ML210 or RSL3. G. Viability curves for WT (Vector) and AGPAT3-targeting sgRNA-expressing 786-O-Cas9 or 769-P-Cas9 cells under indicated concentrations of ML210 or RSL3. H. qRT-PCR analysis of relative AGPAT3 mRNA expression in 786-O cells expressing shNC or AGPAT3-targeting shRNAs. B2M was used as a loading control. Each condition has three biological replicates. Error bars represent ±S.D. Students’ t-test was performed between each shRNA and shNC. *, p<0.05. I. Viability curves for shNC or AGPAT3-targeting shRNA-expressing 786-O cells under indicated concentrations of ML210 or RSL3. J. Viability curves for WT (Vector) and TMEM30A-sgRNA expressing 786-O-Cas9 or 769-P-Cas9 cells under indicated concentrations of ML210 or RSL3. K. Immunoblot analysis of TMEM30A protein levels in WT (EV) and two TMEM30A-targeting sgRNA-expressing 786-O-Cas9 and 769-P-Cas9 cells. β-Actin is used as a loading control. L. Viability time-course of the GPX4 +/+ -786-O-WT, GPX4 −/− -Vector (single knockout, sKO), and double knockout (dKO) lines including GPX4 −/− -ACSL4-sg1 , GPX4 −/− -KEAP 1-sg1, GPX4 −/− -AGPAT3-sg1 , GPX4 −/− -AGPAT3-sg2 , and GPX4 −/− -TMEM30A-sg 1 cells post Fer-1 withdrawal. Viability is relative to the 0-h condition before Fer-1 withdrawal. Representative plot of experiments repeated three times is presented. For viability assays in panel D,F,G,I and J , viability under each condition is relative to that of the respective DMSO-treated condition. Each data point has four biological replicates, and error bars represent ±S.D. Representative plot of experiments repeated three times is presented. See also Figure S2.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Genome Wide, CRISPR, Transduction, Expressing, Infection, Control, Sequencing, Western Blot, Plasmid Preparation, Quantitative RT-PCR, shRNA, Knock-Out, Double Knockout

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. mRNA expression of the indicated genes in ccRCC (N=535; red) or non-ccRCC (N=9,187; grey) tumor samples collected in the TCGA RNA-Seq database. Student’s T-test, ****, p<0.0001; ns, not significant. B. EPAS1 mRNA expression in the indicated cancer types including ccRCC (red) from the TCGA RNA-Seq datasets. Abbreviations are the same as in . C. Immunoblot analysis of HIF-2α expression in ccRCC cell lines OS-RC2, 786-O, 769-P, and RTCC cell line BFTC909. β-Actin was used as a loading control. D. Immunoblot showing HIF-2α and HIF-1β protein levels in wildtype (WT) 786-O cells and four EPAS1 −/− clones generated by CRISPR/Cas9 and single-cell clone isolation. E. qRT-PCR analyses of mRNA levels of HIF-2α target genes CCND1, NDRG1, PLIN2, SLC2A1 (GLUT1), and VEGFA in 786-O WT or the indicated EPAS1 −/− clones. B2M was used as an internal control. Error bars represent ±S.D. F. Immunoblot analysis of HIF-2α and HIF-1β protein levels in wildtype (EPAS1 +/+ ), two EPAS1 −/− single-cell clones 1D7 and 1E3, each expressing an empty vector or EPAS1 -GFP (EPAS1) construct. G. qRT-PCR analyses of mRNA levels of EPAS1 and HIF-2α target genes NDRG1 and VEGFA in 786-O cells expressing shNC or EPAS1-targeting shRNAs. GAPDH was used as an internal control. Error bars represent ±S.D. Student’s T-test, *, p<0.05. H. Immunoblot analysis of NDRG1 and HIF-2α protein expression in 786-O cells expressing shNC or EPAS1-targeting shRNAs. β-Actin was used as a loading control. I. Viability curves for 786-O cells expressing shNC or EPAS1-targeting shRNAs treated with indicated concentrations of RSL3. Viability under each condition is relative to that of the no RSL3 treatment condition. Representative plot of experiments repeated three times. Each data point has four biological replicates and error bars represent ±S.D. J. Chemical structure of HIF-2α antagonist compound 2 (C2). K. qRT-PCR analyses of mRNA levels of HIF-2α target genes NDRG1, VEGFA, CCND1, PLIN2, and SLC2A1 (GLUT1) in 769-P, 786-O-WT and EPAS1 −/− 1D7 cells treated with DMSO or C2 for 3 days. Expression is relative to each of the DMSO-treated WT cells. B2M was used as an internal control. Student’s T-test, *, p<0.05, **, p<0.01, ****, p<0.0001, ns, not significant. L. Immunoblot analysis of NDRG1 and HIF-2α protein expression in 786-O cells treated with DMSO or C2. β-Actin was used as a loading control. M. Viability curves for the indicated cell lines pre-treated with DMSO or C2 for 3 days, then treated together with indicated concentrations of ML210 or RSL3. Viability under each condition is relative to that of the no ML210 or RSL3 treatment condition. Representative plot of experiments repeated three times. Each data point has four biological replicates, and error bars represent ±S.D. N. Scatterplot showing the GPX4 dependency scores (CERES) by CRISPR in cancer cell lines possessing wildtype (WT, N=421; blue) or mutant VHL (N=12; red) in the DepMap database. Mann-Whitney-Wilcoxon test, **, p < 0.01. O. Viability curves for HK-2 cells expressing empty vector or exogenous HIF-2α P405A/P531A , or HIF-2α P405A/P531A/N847A constructs, treated with indicated concentrations of ML210 or RSL3. Viability is relative to that of the DMSO-treated condition. Each data point has four biological replicates, and error bars represent ±S.D. P. Scheme summarizing the lipidomics experiment with 786-O WT, EPAS1 −/− derivative clones 1D1 and 1D7 (1D7-EGFP), and 1D7-EPAS1-GFP cells. Three biological replicates were included for each condition. (S) , ferroptosis-sensitive; (R) , ferroptosis-resistant. Q. Pie chart summarizing the number of lipid species from each class detected in the lipidomic profiling of 786-O WT cells. Abbreviations: CE, cholesterol ester; Cer, ceramide; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ePC, (vinyl ether-linked) PC-plasmalogen; ePE, (vinyl ether-linked) PE-plasmalogen; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. R. Volcano plot highlighting the changes of the indicated lipid classes between the indicated groups. Abbreviations are the same as in panel Q . S. Scheme summarizing the role of HIF-2α in lipid metabolism and ferroptosis susceptibility.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Control, Clone Assay, Generated, CRISPR, Isolation, Quantitative RT-PCR, Plasmid Preparation, Construct, Mutagenesis, MANN-WHITNEY

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Immunoblot showing the HIF-2α and HIF-1β protein levels in wildtype (EV) or EPAS1 -targeting sgRNA-expressing 786-O-Cas9 and 769-P-Cas9 cells. β-Actin is used as a loading control. B. Viability curves of wildtype (Vector) or EPAS1-targeting sgRNA-expressing 786-O-Cas9 or 769-P-Cas9 cells treated with indicated concentrations of ML210 or RSL3. C. Viability curves for WT 786-O or EPAS1 −/− clones treated with indicated concentrations of ML210 or RSL3. D. Viability curves for EPAS1 −/− 786-O single-cell clones 1D7 and 1E3 expressing vector or EPAS1-GFP, then treated with indicated concentrations of ML210 or RSL3. E. Relative viability of EPAS1 +/+ 786-O-Cas9 and EPAS1 −/− 1D7-Cas9 cells transduced with control (sg-GFP, sg-Firefly Luciferase , or sg-LacZ) or GPX4-targeting sgRNAs at day 7 post-infection. Each data point has four biological replicates, and error bars represent ±S.D. Student’s t-test was performed between each of GPX4-targeting sgRNAs and the sg-GFP condition. *, p<0.05, **, p<0.01, ****, p<0.0001, ns, not significant. F. Viability curves for 786-O cells expressing shNC or EPAS1-targeting shRNAs treated with indicated concentrations of ML210. G. Principal component plot of the lipidomic profiles for the indicated cell lines. (S), ferroptosis-sensitive; (R), ferroptosis-resistant. H. Heatmap representing the relative lipid abundances in the indicated cell lines. The abundance of each lipid species is normalized to the mean of that in the EPAS1 +/+ 786-O WT cells and then log2 transformed. The lipids are grouped by classes, and within each class, the lipid species are ordered first with increasing carbon number, then with increasing unsaturation levels. Abbreviations: CE, cholesterol ester; Cer, ceramide; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ePC, (vinyl ether-linked) PC-plasmalogen; ePE, (vinyl ether-linked) PE-plasmalogen; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. Blue: down-regulated relative to the WT cells, red: up-regulated relative to the WT cells. The “wave”-like pattern in the TAG class corresponds to the more significant losses in the polyunsaturated fatty acyl (PUFA)-TAGs than saturated/monounsaturated fatty acyl (SFA/MUFA)-TAGs in response to HIF-2α-depletion. I. Volcano plot showing changes in TAGs grouped as PUFA-TAGs (red fill) and SFA/MUTA-TAGs (white fill) between the indicated cell lines. J. Volcano plot showing changes in PE and ePE lipids grouped as PUFA-PE/ePEs (red fill) and SFA/MUFA-PE/ePEs (white fill) between the indicated cell lines. K. Bar graph representing the relative abundances of the indicated PUFA-PE/ePE lipids in the labeled groups. Log2 fold changes relative to 786-O WT cells are presented for each condition. Error bars represent ±S.D. L. Bar graph representing the relative abundances of the indicated free fatty acids in the labeled groups. nd, not-detectable under the experimental condition. Three biological replicates were included for each condition and error bars represent ±S.D. For viability assays in panel B,C,D and F , viability under each condition is relative to that of the respective DMSO-treated condition. Each data point has four biological replicates, and error bars represent ±S.D. Representative plot of experiments repeated three times is presented. See also Figure S3.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Clone Assay, Transduction, Luciferase, Infection, Transformation Assay, Labeling

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Scheme summarizing the role of ACSL4 and AGPAT3 in PUFA-lipid synthesis, and the potential flux of PUFA-phosphatidic acids (PUFA-PA) towards either PUFA-TAG or PUFA-phospholipid synthesis through distinct enzymatic pathways. B. Volcano plots showing changes in each PE/ePE species grouped as PUFA-PE/ePEs and SFA/MUFA-PE/ePEs between the indicated cell lines. Each condition includes three biological replicates. C. Bar graph showing relative abundances of the PUFA-PE/ePE lipids in 786-O cells expressing control vector or AGPAT3-sg1 . Log2 fold changes between the knockout test cells and the 786-O-Cas9 WT cells were plotted. Error bars represent ±S.D. D. Bar graph showing the relative abundances of the PUFA-PE/ePE lipids in 769-P cells expressing control vector, ACSL4-sg1, or AGPAT3-sg1. E. Bar graph showing the relative abundances of each TAG species in the cell lines indicated. F. Relative changes in the abundances of C50-C60 TAGs in AGPAT3-sg1 or 1 D7-EPAS1 −/− cells versus 786-O-Cas9 WT cells. Error bars represent ±S.D. G. Gene-expression correlation analysis between AGPAT3 and the top 50 RCC-enriched transcription factors including HNF1B (red) in the RNA-Seq dataset of ccRCC tumor samples (N=535) from the TCGA database. Correlation z-scores are plotted using Fisher’s z-transformation on raw correlation coefficients. H. Compound sensitivity-gene-expression correlation analysis in CTRP, highlighting that HNF1B expression is strongly correlated with sensitivity to GPX4 inhibitors ML210, RSL3, and ML162. Negative z-score means high expression is correlated with high sensitivity to compound, and vice versa. I. ChIP-Seq analysis of (data from GSE71250) Hnf-1β binding sites at the Agpat3 and Rassf7 loci in mouse renal mIMCD cells. Track height, reads per million reads (RPM). Bottom: highlight of Hnf-1β consensus motif at the Agpat3 promoter-binding peak center. J. Scheme summarizing xenograft experiments with the indicated cell lines. GPX4 +/+ 786-O-WT, GPX4 −/− (sKO), or GPX4 −/− cells expressing AGPAT3-sgRNA1 and sgRNA2 (dKOs) were subcutaneously implanted into immunocompromised nude mice. Matrigel containing J. 5μM Lip-1 was used to assist the initial implantation of the cancer cells. Additionally, mice bearing GPX4 −/− (sKO) tumors were separated into two groups, with one group treated with vehicle and the other group treated with Lip-1 daily for 10 days. K. Time course of tumor volume measurements of indicated xenografts (five mice per group, two tumors per mouse). Error bars represent ±S.D. Student’s t-test is performed between the volumes of vehicle and Lip-1 treated GPX4 −/− (sKO) tumors at day 14, and between vehicle treated GPX4 −/− (sKO) tumors and GPX4 −/− -AGPAT3-sg1 or GPX4 −/− -AGPAT3-sg2 (dKO) tumors at day 40. ***, p<0.001. See also Figure S6.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Expressing, Control, Plasmid Preparation, Knock-Out, RNA Sequencing Assay, Transformation Assay, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: HIF-2α drives an intrinsic vulnerability to ferroptosis in clear cell renal cell carcinoma

doi: 10.1101/388041

Figure Lengend Snippet: A. Scheme summarizing lipidomic profiling experiments with 769-P-Cas9-vector, ACSL4-sg1 , or AGPAT3-sg1 cells and 786-O-Cas9-vector, AGPAT3-sg1 cells. Each condition includes three biological replicates. (S) , ferroptosis-sensitive; (R) , ferroptosis-resistant. B. Principal component plots for the lipidomic profiles of the 769-P-Cas9-vector, ACSL4-sg1 and AGPAT3-sg1 cells (left), and for the 786-O-Cas9-vector, AGPAT3-sg1 cells (right). C. Heatmap presenting relative lipid abundances in the indicated cell lines. The lipid ratios between ACSL4-sg1, AGPAT3-sg1 and WT of 769-P and 786-O cells were log2 transformed and plotted. Each group includes three biological replicates. Color key: blue: down-regulated in the test cell line, red: up-regulated in the test cell line. Lipid class abbreviations are the same as in . D. Volcano plots showing changes in each TAG species grouped as PUFA-TAGs and SFA/MUFA-TAGs between the indicated cell lines. E. Scheme summarizing the flow of fatty acid metabolism between free PUFA, PUFA-TAG, and PUFA-phospholipids (PUFA-PL) (a), and major lipidomic changes in HIF-2α-depleted (b), or ACSL4 or AGPAT3-depleted (c) cells. Loss of HIF-2α leads to down-regulation of HILPDA/G0S2 and free PUFA levels, which subsequently reduces PUFA-TAG and PUFA-PL deposition (b). When ACSL4 or AGPAT3 is depleted, PUFA-phospholipid synthesis is blocked and free PUFAs are incorporated into the reciprocal PUFA-TAGs (c). F. The ratios between PUFA-PE/ePE and total PE/ePE (left), and between PUFA-PC/ePC and total PC/ePCs in ccRCC tumor samples (N=49; red) and the matched normal tissues (N=49; grey) from previously reported lipidomics datasets. Student’s T-test, ***, p < 0.001. G. Volcano plots showing changes in each PE/ePE species grouped as PUFA-PE/ePEs (red fill) and SFA/MUFA-PE/ePEs (white fill) between high-grade (grade III/IV, N=18) and low-grade (grade I/II, N=16) ccRCC tumor samples. H. AGPAT3 mRNA expression in the indicated cancer types including ccRCC (red) from the TCGA RNA-Seq datasets. Abbreviations are the same as in . I. qRT-PCR analysis of AGPAT3 mRNA levels in ccRCC cell lines 769-P, 786-O, OS-RC2, immortalized normal renal epithelial cell line HK-2 and RTCC cell line BFTC909. Expression relative to B2M was plotted. Representative plot of experiments repeated three times. Each condition includes three biological replicates. Error bars represent ±S.D.

Article Snippet: For CRISPR/Cas9-mediated genome-editing, 786-O and 769-P cells were engineered for Cas9 expression with the pLX-311-Cas9 vector (Addgene 96924), which contains the blasticidin S-resistance gene driven by the SV40 promoter and the SpCas9 gene driven by the EF1 a promoter. sgRNA sequences were cloned into the pLV709 doxycycline-inducible or pXPR_BRD050 constitutive sgRNA expression vectors.

Techniques: Plasmid Preparation, Transformation Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR