Fig 1: TRAIL depletion attenuates ccRCC proliferation in vitro and tumor growth in vivo.A. qRT-PCR result showing relative mRNA expression of TNFSF10 of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 24 hours) conditions (n=3). B. Immunoblotting of TRAIL of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 48 hours) conditions. C. Cell proliferation curves of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml) conditions over a 96-hour period. Each data point was calculated from the cell confluency images (n=6). D. Clonogenic assay showing numbers of colonies (diameter of colony > 0.2cm) of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 10 days) conditions (n=3). E. Spider plots showing normalized bioluminescence imaging (BLI) signals for nude mice orthotopically injected with 786Odish-TRAIL cells and fed with doxycycline diet (Dox on, n=5) and control diet (Dox off, n=4). F. Representative BLI images (day 0 and 35) and normalized BLI signals (day 35) for Dox off and Dox on groups.In A, C, D and F, error bars represent SEM. Unpaired t-test for A, C and D, Mann-Whitney test for F, **P<0.01, ****P<0.0001.
Fig 2: 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.
Fig 3: 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.
Supplier Page from BioLegend for Recombinant Human TRAIL (TNFSF10) (carrier-free)