p53 cdna orf  (Sino Biological)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: In vitro CRISPR screening identified ULK1 as a combinatorial target with an MDM2 inhibitor. A MDM2 mRNA expression (log2(FPKM + 1)) among all paired samples from the TCGA grouped by cancer. Each point represents one sample. The P values are based on two-tailed Student’s t test. B Summary of the correlation between MDM2 expression and overall survival (OS) based on univariate Cox regression and Kaplan‒Meier analyses. Red indicates that MDM2 is a risk factor affecting the prognosis of cancer patients, and green represents protective factors. Only P values < 0.05 are shown. C Schematic diagram of the in vitro screening process used to identify novel drug combinations. D Dot plots showing gene-specific CRISPR viability scores (log fold change and RRA scores). The points ranked in the top ten are highlighted in blue. E Venn diagram showing the intersection of the top 15 genes ranked by the log fold change score and the top 15 genes ranked by the RRA score. F Heatmap of RNA-seq analysis of nine TP53 wild-type cancer cell lines. G Survival curves of APG-115 in nine TP53 wild-type cancer cell lines. H Correlation analysis between gene expression and the IC50 of APG-115. I Western blot showing ULK1 protein levels in A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1. J Cell viability was measured by an MTT assay. A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1 were treated with APG-115 for 72 h

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: In Vitro, CRISPR, Expressing, Two Tailed Test, RNA Sequencing Assay, Western Blot, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: P53 activation combined with ULK1 deficiency can initiate pyroptosis. A Evaluation of APG-115-induced pyroptosis in A2780 sgAAV1 and A2780 sgULK1 cells by phase contrast imaging. B Flow cytometric analysis of Annexin V-FITC and PI staining in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. C LDH release was detected using an LDH Cytotoxicity Detection Kit (Beyotime) in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. **** P < 0.0001. D, F Flow cytometric analysis of FITC staining, PI staining, and LDH release in A549 p53-overexpressing cells following treatment with TNFα + CHX for 24 h. **** P < 0.0001. E, G Flow cytometric analysis of FITC staining, PI staining, and LDH release in A2780 ULK1 knockout cells following treatment with TNFα + CHX for 24 h. H RNA was extracted from the indicated cells, and the expression of GSDMA-E was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. I Western blot showing GSDME protein levels in indicated A2780 cells

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Imaging, Staining, Knock-Out, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: P53 directly activates the transcription of GSDME. A Dot plot showing the differences in the mRNA expression of GSDME between patients with different TP53 mutation statuses. Each point represents one sample. B Bar plot showing the correlation between the mRNA expression of GSDME and that of TP53 in 1210 cell lines from the CCLE database grouped by organ system. C, D p53 affects the expression of GSDME. A2780 and A549 cells were transfected with the indicated plasmids, and the expression of GSDME was determined by immunoblotting. E, F RNA was extracted from the indicated cells, and the expression of GSDME was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. G Illustration of the truncation fragments of the GSDME promoter. H, I Firefly luciferase activity was measured and normalized to that of Renilla luciferase as the internal control. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J, K The published ChIP-seq dataset was reanalyzed via the UCSC Genome Browser. After p53 was activated with MDM2 inhibitors, a peak appeared in the GSDME promoter region. L The indicated HEK-293 T p53-overexpressing cells were subjected to a ChIP assay using an antibody against p53. Isotype-matched IgG was used as a negative control. ** P < 0.01. The data are representative of three independent experiments

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Expressing, Mutagenesis, Transfection, Western Blot, Luciferase, Activity Assay, Control, ChIP-sequencing, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: The synergistic induction of pyroptosis by p53 activation and ULK1 depletion depends on mitochondria quality control. A, B Immunoblot analysis of ULK1, TOMM20, HSPD1, TIM23 and GAPDH expression in indicated cells. The indicated A2780 cells were treated with10 µM APG-115 for 24 h. C Immunofluorescence images of A2780 cells with deletion of ULK1 and treatment with 10 µM APG-115 for 24 h. The cells were costained with MitoTracker Deep Red and DAPI. The white line represents 10 µm. D, E Flow cytometric analysis of FITC- and PI-stained control, siFUNDC1, and siPARK A2780 cells treated with 10 µM APG-115 for 24 h. F , G Survival fractions of control, siFUNDC1, and siPARK A2780 cells following treatment with 10 µM APG-115 for 24 h. H, I LDH release was detected in models of mitophagy deficiency or macroautophagy deficiency following treatment with 10 µM APG-115 for 24 h. ns: P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Schematic of mechanisms underlying the synergy between MDM2i and ULK1 deficiency in TP53 wild-type cells. See main text for a detailed description

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: Combined targeting of mitophagy and activation of p53 could be used to reverse platinum resistance. A GSVA scores of mitophagy-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. The sensitivity of the tumor samples was calculated using the "pRRophetic" R package, and the optimal cutoff value of the ROC curve was selected as the cutoff point between sensitivity and resistance based on the Youden index. The P values were calculated using the Wilcoxon rank–sum test. B GSVA scores of pyroptosis-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. C Illustration showing the process for generating cisplatin-resistant cell lines. D Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h. E Immunoblotting of ULK1, TOMM20, HSPD1, TIM23 and GAPDH in extracts of cisplatin-resistant cells. F, G GSDME expression in cisplatin-resistant cells. *** P < 0.001. H, I The indicated cisplatin-resistant cells were treated with TNFα + CHX for 24 h. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

doi: 10.1186/s13046-024-03168-8

Figure Lengend Snippet: Schematic of ULK1 deficiency and p53 activation promote pyroptosis by activating GSDME directly (By Figdraw)

Article Snippet: The human full-length TP53 cDNA (HG10182-G, Sino Biological, Beijing, China) was cloned and inserted into the pSIN-EF2-puro vector (P40791, MiaoLingBio, Wuhan, China) between the BamHI and EcoRI restriction sites.

Techniques: Activation Assay

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) Immunoblots of STING in CD4+Cas9+ T cells transduced with sgRNAs targeting STING and p53 at 4 days after retroviral infection. Actin was used as loading control. Representative blots (left) and quantification (right) represented as the mean ± s.e.m. of n = 3 mice and 2 independent experiments. (b) CD80 expression in CD4+Cas9+ T cells transduced with sgRNAs targeting STING and p53, restimulated for 2 days with anti-CD3+28 dynabeads and treated or not (vehicle) with 3 μg/ml DMXAA. Representative flow cytometry plots (left) and quantification (right). Data represent the mean ± s.e.m. of at least 3 mice, pooled from 2 independent experiments. (c) CFSE dilution in CD4+Cas9+ T cells transduced with sgRNAs targeting STING and p53, restimulated for 3 days with anti-CD3+28 dynabeads and treated or not (vehicle) with STING agonists and idasanutlin (nutlin). Representative flow cytometry plots (left) and quantification (right) of the frequency of proliferating cells. Data represent the mean ± s.e.m. of 3–5 mice, pooled from 2 independent experiments. Statistical analysis by two-tailed, unpaired Student’s t test (a,b) and 2-way ANOVA with Dunnett’s multiple comparisons test (c). Not significant (n.s.) P > 0.05, *P<0.05, **P<0.01 and ***P<0.001. #P<0.001 between treatment vs vehicle untreated.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Western Blot, Transduction, Infection, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a-c) Immunoblots of total and phospho-STING (S366) protein expression in WT CD4+ T cells activated for 2 days with anti-CD3+28 and treated or not with the STING inhibitor H-151. Cell were stimulated with 10 μg/ml 2’3’cGAMP (a), 5 μg/ml c-di-AMP (b) or 3 μg/ml DMXAA (c) for 3–6 hours. Actin was used as loading control. Representative blots (left) and quantification (right) of at least 3 independent experiments and 4, 3, and 5 mice for 2’3’cGAMP, c-di-AMP, and DMXAA treatment, respectively. (d-g) Flow cytometry analysis of Ki67 expression (d), apoptosis measured by annexin V and active caspase (e), p53 (f), and CD80 expression (g) in WT CD4+ T cells stimulated for 3 days with anti-CD3+28 and treated or not with STING agonists and pre-treated or not with the STING inhibitor H-151. Representative flow cytometry plots (left) and quantification (right) of at least 6 mice per treatment, pooled from 4–6 independent experiments and shown as mean ± s.e.m. (h) Correlation analysis of CD80 and p53 expression in T cells stimulated and treated as shown in (f,g). Statistical analysis in (a-g) by two-tailed, unpaired Student’s t test. **P<0.01 and ***P<0.001. ##P < 0.01 and #P<0.001 between STING agonists vs. vehicle untreated.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Inhibition, Expressing, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) Differentially expressed genes (DEG) between wild-type and Lrrc8c−/− CD4+ T cells before and after anti-CD3+CD28 stimulation and analyzed by RNA-seq. (b) Scatterplot of fold-change in gene expression (log2 FC) vs. adjusted p-value (−Log10) between wild-type and Lrrc8c−/− CD4+ T cells after anti-CD3+CD28 stimulation. Blue and red dots indicate downregulated and upregulated genes in Lrrc8c−/− T cells, respectively. Green dots indicate DEGs belonging to the p53 pathway. (c) IPA and KEGG pathways of DEGs in stimulated Lrrc8c−/− CD4+ T cells and ranked by P-value. (d) Top-5 upstream regulators that are inhibited (blue) or activated (red) in Lrrc8c−/− compared to wild-type CD4+ T cells after anti-CD3+CD28 stimulation and ranked by activation z-score. (e) GSEA showing enrichment in p53 pathway genes comparing transcriptomes of Lrrc8c−/− and wild-type CD4+ T cells after anti-CD3+CD28 stimulation. (f) Heat map of DEGs associated with the p53 pathway identified by GSEA in (e). Relative mRNA expression/row (red, high; blue, low). (g) mRNA expression of proapoptotic and cell cycle arrest genes in wild-type and Lrrc8c−/− CD4+ T cells before and after stimulation with anti-CD3+CD28 and measured by RT-qPCR. Rlp32 mRNA was used as housekeeping control (n=4 mice per genotype). (h) Immunoblot of total and phosphorylated p53 (p-p53 S15) in wild-type and Lrrc8c−/− CD4+ T cells before and after stimulation with anti-CD3+CD28. Actin was used as loading control. (i) Quantification of p53 expression in wild-type and Lrrc8c−/− CD4+ T cells stimulated for 3 days and normalized to actin loading control. Data are representative (h) and averaged (i) from n=5 mice/genotype and pooled from 2 independent experiments. (j) Cell cycle analysis of wild-type and Lrrc8c−/− CD4+ T cells before and after stimulation with anti-CD3+CD28. Representative flow cytometry plots (left) and quantification (right) of different cell cycle phases (n=6 and 8 mice/genotype for unstimulated and stimulated conditions, respectively). (k) CFSE dilution in wild-type and Lrrc8c−/− CD4+ T cells stimulated with anti-CD3+CD28. (l) Fold-change of CD4+ T cell numbers from (k) 3 days after anti-CD3+CD28 stimulation (compared to day 1, n=8 mice/genotype). (m) Ki67 expression in wild-type and Lrrc8c−/− CD4+ T cells before and after stimulation with anti-CD3+CD28. Representative flow cytometry plots (left) and quantification (right) of Ki67+ cells (n=9 mice/genotype, pooled from 3 independent experiments). All data are mean ± s.e.m. and were analyzed by two-tailed, unpaired Student’s t test. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: RNA Sequencing Assay, Expressing, Activation Assay, Quantitative RT-PCR, Western Blot, Cell Cycle Assay, Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) CD80 mRNA expression in human T cells from healthy volunteers stimulated with phytohemagglutinin for 3 days and treated with nutlin-3 for 24h. CD80 mRNA expression based on microarray data from GSE110369 (Ref62). (b) p53 binding to the CD80 gene locus in human T cells treated as in (a) and analyzed by ChIP-Seq (data source: GSE110368)62. Representative binding peaks in the CD80 promoter (left) and quantification (right). (c) CD80 expression in CD4+Cas9+ T cells transduced with sgRNAs targeting p53 (Trp53) and restimulated for 3 days with anti-CD3+28 and treated or not with nutlin. Representative flow cytometry plots (left) and quantification (right) of mean fluorescence intensities (MFI) of CD80. Data are from 3 and 5 mice for sgControl and sgTrp53, respectively, pooled from 2 independent experiments. (d) Cd80 mRNA expression in CD4+ T cells of WT or Lrrc8c−/− mice stimulated with anti-CD3/28 for 1 and 2 days. T cells from WT mice were treated or not with DCPIB for the duration of T cell stimulation. mRNA expression based on RNA-Seq data (compare with Fig. 3). Data are from 3 mice per genotype and treatment. (e) CD80 expression in WT and Lrrc8c−/− CD4+ T cells before and after stimulation with anti-CD3+28 for 3 days. Representative flow cytometry plots (left) and quantification (right) from 11 mice per genotype, pooled from 4 independent experiments. (f) CD80 expression in WT CD4+ T cells treated with 20 μM DCPIB for 3 days following anti-CD3+28 stimulation. Representative overlay histograms (left) and quantification (right) of CD80 expression from 6 mice per condition. (g,h) CD80 cell expression in WT and Lrrc8c−/− CD4+ T cells upon stimulation with anti-CD3+28 for 3 days and treated or not with nutlin (in g), or after 3 days of retroviral transduction with empty vector or p53 (in h). Representative flow cytometry plots (left) and quantification (right) of 8 mice per genotype, pooled from 4 and 2 independent experiments in (g) and (h), respectively. All data are mean ± s.e.m. and were analyzed by two-tailed, unpaired Student’s t test (a-c,e,g,h) and by two-tailed, paired t test (f). Not significant (n.s.) P > 0.05, *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Expressing, Microarray, Binding Assay, ChIP-sequencing, Transduction, Flow Cytometry, Fluorescence, Cell Stimulation, RNA Sequencing Assay, Plasmid Preparation, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) Immunoblots of total p53 expression in wild-type and Lrrc8c−/− CD4+ T cells after stimulation with anti-CD3+CD28 for 3 days in the presence and absence of the MDM2 antagonist idasanutlin (abbreviated as nutlin). Actin was used as loading control. Representative blots (left) and quantification (right) from 4 independent experiments and 8 mice per genotype and treatment. (b) Cell cycle analysis of wild-type and Lrrc8c−/− CD4+ T cells after stimulation for 3 days in the presence or absence of nutlin. Representative flow cytometry plots (lefts) and quantification (right) of the different cell cycle phases. Data are from 12 mice per genotype and treatment, and pooled from 5 independent experiments. (c) Ki67 expression in wild-type and Lrrc8c−/− CD4+ T cells stimulated for 3 days and treated or not with nutlin. Representative flow cytometry plots (left) and quantification (right) of Ki67+ cells from 12 mice per genotype pooled from 5 independent experiments. (d) Apoptotic T cells from wild-type and Lrrc8c−/− mice measured by annexin V and active caspase (VAD-FMK) staining 3 days after stimulation and treated or not with nutlin. Representative contour plots (left) and quantification (right) of apoptotic cells from 14 mice per genotype pooled from 5 independent experiments. All data are mean ± s.e.m. and were analyzed by two-tailed, unpaired Student’s t test. **P < 0.01 and ***P < 0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Western Blot, Expressing, Cell Cycle Assay, Flow Cytometry, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a-c) Protein expression of p53 (a), Ki67 (b), and apoptosis determined by annexin V staining (c) in WT and Lrrc8c−/− CD4+ T cells after 3 days of retroviral transduction. Representative flow cytometry plots (left) and quantification (right). Data represent the mean ± s.e.m. of 8 mice per genotype, pooled from 2 independent experiments. (d,e) Protein expression of p53 (d) and Ki67 (e) in CD4+Cas9+ T cells transduced with sgRNAs targeting p53 (Trp53) after 3 days of retroviral infection. Representative flow cytometry plots (left) and quantification (right). Data represent the mean ± s.e.m. of 3 (in d) and 5 (in e) mice, pooled from 2 independents. (f,g) Protein expression of p53 (f) and Ki67 (g) in T cells shown in (d,e) after restimulation for additional 3 days with anti-CD3+28 dynabeads and treated or not with idasanutlin (nutlin). Representative flow cytometry plots (left) and quantification (right). Data represent the mean ± s.e.m. of 3 and 5 mouse donors for sgControl and sgTrp53, respectively, pooled from 2 independents. Statistical analysis by two-tailed, unpaired Student’s t test. *P<0.05 and ***P<0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Expressing, Staining, Transduction, Flow Cytometry, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) Averaged traces (left) and quantification (right) of RVD (AUC) in CD4+ T cells stimulated with anti-CD3+28 for at least 3 days and subjected to hypotonic solution after treatment for 30 min with 20 μM DCPIB (data are the mean ± s.e.m. of 12 and 20 traces for vehicle and DCPIB treated cells, respectively, pooled from at least 5 independent experiments). (b) IVRAC traces from CD4+ T cells stimulated for 2d with anti-CD3+28 and pre-treated or not with 20 μM DCPIB, measured by patch clamping in whole-cell configuration. Representative traces from 10 cells (vehicle) and 5 cells (DCPIB) from at least 3 independent experiments. Recording protocol (top): T cells were held at −70 mV and were depolarized to +80mV every 5s in hypotonic solution (~215 mOsm). (c) GSEA of RNA-Seq data from WT CD4+ T cells treated or not with 20 μM DCPIB identifies DEGs associated with p53 pathway after anti-CD3+28 stimulation. (d) Venn-diagram showing number of DEGs related to the p53 pathway between WT vs Lrrc8c−/− and WT vs WT + DCPIB CD4+ T cells after anti-CD3+28 stimulation for 1 and 2 days. (e) Heat map of DEGs associated with p53 pathway identified by GSEA in (c). Color coding: high (red) and low (blue) relative mRNA expression per row. DEGs highlighted in red are shared between WT vs WT + DCPIB and WT vs Lrrc8c−/− CD4+ T cells. Statistical analysis in (a) by two-tailed, unpaired Student’s t test. ***P<0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: RNA Sequencing Assay, Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a,b) Cytosolic Ca2+ signals in WT T cells stimulated for 3 days with anti-CD3+28 and treated or not with the STING inhibitor H-151 and STING agonists. T cells were stimulated with thapsigargin (TG) in Ca2+-containing Ringer buffer. Averaged Ca2+ traces (a) and area under the curve (AUC) following TG treatment (b). Data are the mean ± s.e.m. of 6 mice (vehicle, DMXAA, 2’3’cGAMP, 3’3cGAMP) or 4 mice (c-di-AMP, c-di-GMP) pooled from 2–3 independent experiments. (c) Cytosolic Ca2+ signals in WT and Lrrc8c−/− T cells 3 days after retroviral transduction with p53 using a similar protocol as in (a). Averaged Ca2+ traces (left) and quantification of the AUC (right) following TG treatment. Data are the mean ± s.e.m. of 4 mice per genotype and treatment, pooled from 2 independent experiments. (d) Cytosolic Ca2+ signals in CD4+Cas9+ T cells transduced with sgRNAs targeting STING and p53 three days after retroviral transduction using a similar protocol as in (a). Averaged Ca2+ traces (left) and quantification of the AUC (right) after TG treatment. Data are the mean ± s.e.m. of 3 mice pooled from 2 independent experiments. Statistical analysis by two-tailed, unpaired Student’s t test. Not significant (n.s.) P > 0.05, **P<0.01 and ***P<0.001. #P<0.001 between treatment vs vehicle untreated.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Transduction, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a) Compound screening to identify substrates of LRRC8C in T cells (Created with BioRender.com). Correlation of CD80 expression and CFSE dilution in wild-type and Lrrc8c−/− CD4+ T cells stimulated for 2 days with anti-CD3/CD28 and treated with different substrates of VRAC channels. Arrows connect wild-type and Lrrc8c−/− T cell samples treated with the same compound at high (thick line) and low (thin line) compound concentrations (n= 4 mice/ genotype, pooled from 2 independent experiments). (b,c) Wild-type and Lrrc8c−/− CD4+ T cells stimulated with anti-CD3/CD28 were treated with increasing concentrations of CDNs and analyzed for cell proliferation and CD80 expression. Graphs show the differences in proliferation (Δ%CFSElow cells, in b) and the differences in CD80 expression (DMFI, in c) between at least three CDN concentrations (Δ[CDN]). Compare with Extended Data Fig. 7c,​,dd (n=6 mice/genotype and treatment, pooled from 3 independent experiments). (d) Intracellular concentration of cGAMP in T cells exposed or not to 5 μg/ml 2’3’cGAMP in hypotonic buffer (~215 mOsm) for 15 min and measured by ELISA (n=10 mice/genotype, pooled from 2 independent experiments). (e) Immunoblots of total and phosphorylated STING (p-STING S366) in wild-type and Lrrc8c−/− CD4+ T cells after treatment with 10 μg/ml 2’3’cGAMP for 6h. Actin was used as loading control. Representative blots (left) and quantification (right) from n=3 mice/genotype and 2 independent experiments. (f) 2’3’cGAMP amount in culture media collected after in vitro stimulation of CD4+ T cells with anti-CD3+CD28 for 1–3 days measured by ELISA (n=6 mice/genotype, pooled from 5 independent experiments). (g) GSEA of RNA-Seq data identifies DEGs associated with IFN-α response in stimulated wild-type but not Lrrc8c−/− CD4+ T cells. (h,i) Flow cytometry analysis of p53 (h) and CD80 expression (i) in wild-type and Lrrc8c−/− CD4+ T cells stimulated with anti-CD3+CD28 and treated or not with STING agonists. Representative flow cytometry plots (left) and quantification (right). Data are from n=6 mice/genotype, pooled from 3 independent experiments. (j-l) Flow cytometry analysis of p53 (j), CD80 (k), and Ki67 (l) expression in wild-type CD4+ T cells stimulated for 3 days with anti-CD3+CD28 and treated or not with H-151 and idasanutlin (nutlin). Representative flow cytometry plots (left) and quantification (right) from n=6 mice/treatment, pooled from 3 independent experiments. All data are mean ± s.e.m. and were analyzed by two-tailed, unpaired Student’s t test. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, In Vitro, RNA Sequencing Assay, Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: The volume regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling

doi: 10.1038/s41590-021-01105-x

Figure Lengend Snippet: (a,b) Cytosolic Ca2+ signals in naïve (a) and activated (b) CD4+ T cells isolated from wild-type and Lrrc8c−/− mice. Fura-2-loaded T cells were stimulated by anti-CD3 cross-linking in 2 mM Ca2+ containing Ringer solution followed by ionomycin (Iono) stimulation (in a) or thapsigargin (TG) in Ca2+-free buffer followed by re-addition of extracellular Ca2+ (in b). Averaged Ca2+ traces (left) and quantification of the area under the curve (AUC, right) in the regions indicated by the dotted lines (n=9 mice/genotype, pooled from 3 independent experiments). (c) Plasma membrane potential (Vm) in wild-type and Lrrc8c−/− CD4+ T cells activated with CD3+CD28 and measured by patch-clamping. Gigaohm seals were stablished in isotonic solution in voltage-clamp configuration and cells were treated with TG for at least 5 min before recording Vm (n=8 cells/genotype, pooled from 2 independent experiments). (d,e) Cytosolic Ca2+ signals in wild-type and Lrrc8c−/− T cells stimulated with anti-CD3+CD28 and treated or not with STING agonists. T cells were stimulated with TG in Ca2+-containing Ringer buffer. Averaged Ca2+ traces (d) and quantification of the AUC (e) in the regions indicated by the dotted lines (n=10 mice/genotype, pooled from 5 independent experiments). (f) Cytosolic Ca2+ signals in T cells treated or not with STING inhibitor H-151 and idasanutlin (abbreviated as nutlin) using a similar protocol as in (d). Averaged Ca2+ traces (left) and quantification of the AUC (right) in the regions indicated by the dotted lines (n=6 mice/treatment, pooled from 3 independent experiments). (g) Cytosolic Ca2+ signals in wild-type and Lrrc8c−/− T cells treated or not with nutlin using a similar protocol as in (d and f). Averaged Ca2+ traces (left) and quantification of the AUC (right) in the regions indicated by the dotted lines (n=10 mice/treatment, pooled from 5 independent experiments). (h) IL-2 and IFN-γ production by wild-type and Lrrc8c−/− CD4+ T cells activated with CD3+CD28 and re-stimulated for 6h with PMA+Iono. Representative contour plots (left) and quantification (right) of IL-2+ and IFN-γ+ CD4+ T cells (n=10 mice per genotype, pooled from 6 independent experiments). (i) Schematic representation of LRRC8C regulating Ca2+ signals in T cells. CDNs influx via LRRC8C leads to STING activation and p53 stabilization, which in turn suppresses Ca2+ signals in T cells (Created with BioRender.com). All data are mean ± s.e.m. and were analyzed by two-tailed, unpaired Student’s t test. **P < 0.01 and ***P < 0.001.

Article Snippet: For overexpression of murine LRRC8C (Dharmacon, MMM1013–202767571) and p53 (Sino Biological, MG50534-UT), cDNAs were subcloned into the pMSCV retroviral vector (Addgene, 52114); empty pMSCV was used as control.

Techniques: Isolation, Activation Assay, Two Tailed Test