Journal: bioRxiv

Article Title: Synthetic essentiality of TRAIL/TNFSF10 in VHL-deficient renal cell carcinoma

doi: 10.1101/2025.05.29.621197

Figure Lengend Snippet: TRAIL is a HIF2α target and a candidate synthetic essential gene. A. Venn diagram between HIF2α-upregulated genes (logFC(Ctrl/sgEPAS1) > 1.5, FDR < 0.05) and DepMap preferentially essential genes (gene effect < 0, Z-score < -1) in 786O. B. Bubble plot of the 57 overlapped genes. The bubbles’ size and color reflect the false discovery rate (FDR) and Z-score, respectively. TRAIL has lowest Z-score and is highlighted. C. Relative mRNA expression of EPAS1 and TNFSF10 in control and HIF2α-knockout 786O sublines (n=3), measured with qRT-PCR. D. Immunoblotting of HIF2α and TRAIL in control and HIF2α-knockout 786O sublines. E. Relative mRNA expression of TNFSF10 in normal (n=161) and tumor tissues (n=532) of TCGA KIRC patient’s cohort. F. TRAIL protein expression in normal (n=84) and tumor tissues (n=110) of CPTAC KIRC patient’s cohort. G. Scatter plot of TNFSF10 and EPAS1 levels in the TCGA KIRC cohort. Spearman correlation coefficient ρ and P value are marked. H. HIF2α and input ChIP-Seq signals in the TNFSF10 locus with the promoter region chr3:172,242,513 - 172,243,623 highlighted in the red box. I. ChIP-qPCR result for IgG and anti-HIF2α at the TNFSF10 promoter (n=3). In C and I , data represent mean ± SEM. Unpaired t-test for C and I , Mann-Whitney test for E and F , **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Primary antibodies included: TRAIL (Cell Signaling Technology, #3219), HIF2α (Cell Signaling Technology, #59973), p38 (Cell Signaling Technology, #9212), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, #4511), RB (Cell Signaling Technology, #9309), Phospho-RB (Ser780)(Cell Signaling Technology, #8180), cyclin D1(Cell Signaling Technology,#2978P), cyclin D3 (Cell Signaling Technology, #2936), cyclin E1 (Cell Signaling Technology,#4219), E2F1 (Novus Biologicals, #NBP2-37474), Phospho-IκBα (Cell Signaling Technology, #2859), IκBα (Cell Signaling Technology, #4814), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), p-ERK (Cell Signaling Technology, #4370P), ERK (Cell Signaling Technology, #4695). β-actin (Santa Cruz, #sc-47778), Vinculin (Sigma-Aldrich, #05-386).

Techniques: Expressing, Control, Knock-Out, Quantitative RT-PCR, Western Blot, ChIP-sequencing, ChIP-qPCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: Synthetic essentiality of TRAIL/TNFSF10 in VHL-deficient renal cell carcinoma

doi: 10.1101/2025.05.29.621197

Figure Lengend Snippet: TRAIL activates p38 MAPK and facilitates G1/S progression in ccRCC cells. A. Heatmap showing differential gene expression (|fold Change| > 1.5 and FDR < 0.05) between 786O-dishTRAIL treated with doxycycline (Dox on, 48 hours) and untreated controls (Dox off). TNFSF10 is among the downregulated genes by TRAIL knockdown. B. GSEA result showing MSigDB hallmark pathways (P < 0.05, FDR < 0.25) enriched to Dox-off condition. C. Representative flow cytometric plots of annexin-V-APC and DAPI staining on 786O-dishTRAIL under Dox on (48 hours) and Dox off conditions (n=4) and qualification of the percentage of apoptotic cells (red box). D-E. Representative flow cytometric plots for DNA content analysis using propidium iodide (PI) staining of 786O-dishTRAIL under Dox on (48 hours) and Dox off conditions (n=3), based on which the proportions of three cell cycle phases were quantitated. F. Immunoblotting showing changes of the indicated proteins and phosphorylations for 786O-dishTRAIL under Dox on and Dox off conditions for various durations (48, 72, 96 hours). G. Scatter plot of protein levels of TRAIL and p38 in ccRCC CPTAC data. Pearson correlation coefficient ρ and P value are indicated. H. Immunoblotting of p38 and phorpho-p38(Thr180/Tyr182) for 786O-dishTRAIL treated with 10µM necrostatin-1s (Nec-1s) for 1 hour or 48 hours of doxycycline. In C and E , error bars represent SEM. Unpaired t-test, *P < 0.05, ***P < 0.001, # P > 0.05.

Article Snippet: Primary antibodies included: TRAIL (Cell Signaling Technology, #3219), HIF2α (Cell Signaling Technology, #59973), p38 (Cell Signaling Technology, #9212), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, #4511), RB (Cell Signaling Technology, #9309), Phospho-RB (Ser780)(Cell Signaling Technology, #8180), cyclin D1(Cell Signaling Technology,#2978P), cyclin D3 (Cell Signaling Technology, #2936), cyclin E1 (Cell Signaling Technology,#4219), E2F1 (Novus Biologicals, #NBP2-37474), Phospho-IκBα (Cell Signaling Technology, #2859), IκBα (Cell Signaling Technology, #4814), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), p-ERK (Cell Signaling Technology, #4370P), ERK (Cell Signaling Technology, #4695). β-actin (Santa Cruz, #sc-47778), Vinculin (Sigma-Aldrich, #05-386).

Techniques: Gene Expression, Knockdown, Staining, Western Blot

Journal: bioRxiv

Article Title: Synthetic essentiality of TRAIL/TNFSF10 in VHL-deficient renal cell carcinoma

doi: 10.1101/2025.05.29.621197

Figure Lengend Snippet: TRAIL is required to sustain HIF2α expression and activity in ccRCC cells. A. Immunoblotting of HIF2α for 786O-dishTRAIL treated with doxycycline (Dox on) and untreated controls (Dox off) across three time points (48,72, and 96 hours). B. qRT-PCR result of relative mRNA expression of VEGFA and SLC2A1 for 786O-Ctrl, 786O-sgEPAS1, and 786O-dishTRAIL under Dox off and Dox on (96 hours) conditions (n=3). C. Immunoblotting of HIF2α for 786O treated with 25μM SB203580 for 1 hour or 6 hours. D. qRT-PCR result of relative mRNA expression of VEGFA , SLC2A1 and EPAS1 for 786O treated with 25μM SB203580 for 6 hours (n=3). In B and D , error bars represent SEM. Unpaired t-test, ***P < 0.001, # P > 0.05.

Article Snippet: Primary antibodies included: TRAIL (Cell Signaling Technology, #3219), HIF2α (Cell Signaling Technology, #59973), p38 (Cell Signaling Technology, #9212), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, #4511), RB (Cell Signaling Technology, #9309), Phospho-RB (Ser780)(Cell Signaling Technology, #8180), cyclin D1(Cell Signaling Technology,#2978P), cyclin D3 (Cell Signaling Technology, #2936), cyclin E1 (Cell Signaling Technology,#4219), E2F1 (Novus Biologicals, #NBP2-37474), Phospho-IκBα (Cell Signaling Technology, #2859), IκBα (Cell Signaling Technology, #4814), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), p-ERK (Cell Signaling Technology, #4370P), ERK (Cell Signaling Technology, #4695). β-actin (Santa Cruz, #sc-47778), Vinculin (Sigma-Aldrich, #05-386).

Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Synthetic essentiality of TRAIL/TNFSF10 in VHL-deficient renal cell carcinoma

doi: 10.1101/2025.05.29.621197

Figure Lengend Snippet: HIF2α inhibitor synergizes with recombinant TRAIL to reduce 786O viability. A. Normalized percentages of viable 786O-dishTRAIL cells treated with doxycycline and untreated control (48 hours) followed by treatment with two different concentrations of rTRAIL for 24 hours. Error bars represent SEM. Ordinary one-way ANOVA test with Tukey’s multiple comparison correction, ***P < 0.001, ****P < 0.0001. B. Immunoblotting of HIF2α, TRAIL, p-p38, and p38 in 786O treated with 2μM or 4μM belzutifan for 48 hours. C. Cell viability quantification of 786O under three treatments: incremental doses of rTRAIL (black), incremental doses of belzutifan (red), and combinations of rTRAIL and belzutifan at a fix ratio of 100 (cyan). The results were analyzed with CompuSyn software to construct FA-CI plot (right). CI<1 indicates synergism effect.

Article Snippet: Primary antibodies included: TRAIL (Cell Signaling Technology, #3219), HIF2α (Cell Signaling Technology, #59973), p38 (Cell Signaling Technology, #9212), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, #4511), RB (Cell Signaling Technology, #9309), Phospho-RB (Ser780)(Cell Signaling Technology, #8180), cyclin D1(Cell Signaling Technology,#2978P), cyclin D3 (Cell Signaling Technology, #2936), cyclin E1 (Cell Signaling Technology,#4219), E2F1 (Novus Biologicals, #NBP2-37474), Phospho-IκBα (Cell Signaling Technology, #2859), IκBα (Cell Signaling Technology, #4814), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), p-ERK (Cell Signaling Technology, #4370P), ERK (Cell Signaling Technology, #4695). β-actin (Santa Cruz, #sc-47778), Vinculin (Sigma-Aldrich, #05-386).

Techniques: Recombinant, Control, Comparison, Western Blot, Software, Construct