SET1B depletion overcomes resistance to HIF2 inhibition in ccRCC. A, Immunoblots of WT, HIF2α KO, and HIF2α KO 786-0 cells reconstituted with WT HIF2α or the HIF2α G323E mutation. B, HIF2α KO cells reconstituted with WT and HIF2 G323E mutation were treated with 1 µmol/L PT2385 for 24 hours. Endogenous HIF1β was immunoprecipitated, and the interaction with HIF2α was assessed by immunoblotting. Immunoblot is representative of 3 independent experiments. C, HIF2α KO cells reconstituted with WT and HIF2 G323E mutation were treated with 1 μmol/L PT2385 for 24 hours. HIF2α target gene and SET1B mRNA expression was assessed by qPCR (n = 3 biological replicates). Graphs show mean ± SD. Two-way ANOVA. D, HIF2α KO cells reconstituted with WT and HIF2α G323E mutation were transfected with an SET1B siRNA for 48 hours. Twenty-four hours before harvesting, cells were treated with and without 1 μmol/L PT2385. qPCR analysis was performed using primers targeting the VEGFA and ANGPTL4 mRNAs (n = 3 biological replicates). Graphs show mean ± SD. Two-way ANOVA. E, ChIP of H3K4me3 and RNA Pol II (F) in HIF2α KO cells reconstituted with WT and HIF2α G323E mutation following CRISPR-mediated SET1B depletion 24 hours before harvesting cells were treated with 1 μmol/L PT2385. ChIP-PCR was performed using primers targeting the VEGFA promoter (n = 3 biological replicates). Graphs show mean ± SD. Two-way ANOVA. G, ChIP of HIF2α in HIF2α KO cells reconstituted with WT and HIF2α G323E mutation following CRISPR-mediated SET1B depletion 24 hours before harvesting cells were treated with 1 μmol/L PT2385. ChIP-PCR was performed using primers targeting the VEGFA promoter and exon 3 (n = 3 biological replicates). Two-way ANOVA. H, ccRCC mouse xenograft model. WT and SET1B-depleted 786-0 cells expressing luciferase were injected into the tail vein of nude mice. Bioluminescence was measured and quantified from the lungs on day 18, and luminescence was normalized to the vehicle from the specific genotype (control = 6; control + PT2385 = 6; SET1B KO = 6; and SET1B KO + PT2385 = 7). Mean ± SEM. Kruskal–Wallis test). siSET1B, SET1B siRNA.