Fig. 6

YWHAH recruits circEHD2 and YWHAH to the promoter of SOX9. A and B, Co-IP assay showed that YAP could be precipitated by anti-YWHAH antibody (A) and YWHAH could be precipitated by anti-YAP antibody (B) in OSRC-2 cells. C and D, Co-IP assay confirmed that YWHAH could bind to YAP by anti-YWHAH antibody (C) and anti-YAP antibody (D) in the nuclear extracts of OSRC-2 cells. E, Subcellular co-localization of YWHAH and YAP in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. F, qRT–PCR analysis of the level of YAP in RCC cells when cocultured with VP at different doses. G and H, qRT–PCR analysis (G) and western blot assay (H) showed the expression level of SOX9 in OSRC-2 cells when cocultured with VP. I, Schematic illustration of the binding sites of circEHD2 and YAP on SOX9 promoter. J, Schematic illustration of PCR-amplified fragments of SOX9 promoter. K, ChIRP analysis the enrichment of SOX9 promoter fragments in OSRC-2 cells. L, Dual-luciferase reporter assays showed that circEHD2 could bind to the P2 region (-1501-nt to -1001-nt) of the SOX9 promoter. M–P, ChIP-qPCR of YAP (M and O) and YWHAH (N and P) revealed the enrichment of SOX9 promoter in OSRC-2 cells with circEHD2 knockdown (M and N) or overexpression (O and P). Error bars represent the standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001