Fig. 5: DMF-HNF1B axis suppresses YAP activity.

A Co-IP of exogenous Flag-HNF1B and endogenous YAP in HEK-293T cells. B The effect of DMF on the interaction of Flag-HNF1B and YAP was performed by co-IP and western blot. C The effect of overexpressed HNF1B on the interaction between YAP and TEAD4. HEK-293T cells with or without overexpression of Flag-HNF1B were transfected with HA-YAP and Flag-TEAD4 and subjected to Co-IP. D Western blot analysis of pYAP in ccRCC cells with or without DMF treatment. E Succination of endougenous YAP was analyzed by co-IP followed by western blot in 786-O cells with or without DMF treatment. F Western blot analysis of YAP in ccRCC cells with or without DMF treatment. G qPCR analysis of YAP mRNA levels in 786-O and RCC4 cells treated with DMF. The data represent the mean ± SD of n = 3 independent experiments. H Immunofluorescence staining assay of YAP expression in 786-O and RCC4 cells. Red, YAP, blue, DAPI. Scale bars, 20 μm. qPCR analysis of relative CYR61 (I) and AXL (J) mRNA level derived from ccRCC cells treated with or without DMF. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined using Student’s t-test. ***P < 0.001, ****P < 0.0001. K Western blot analysis of YAP in HNF1B KO 786-O cells. qPCR analysis of relative CYR61 (L) and AXL (M) mRNA level derived from HNF1B KO 786-O cells. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined using ordinary one-way ANOVA. **P < 0.01, ***P < 0.001. N qPCR analysis of MYC mRNA level derived from786-O and RCC4 cells treated with or without DMF. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined using Student’s t-test. **P < 0.01, ***P < 0.001. O qPCR analysis of MYC mRNA levels in control and HNF1B KO 786-O cells. The data represent the mean ± SD of n = 3 independent experiments. P Western blot analysis of YAP levels in 786-O and RCC4 cells treated with DMF and transfected with Flag-HNF1B as indicated. Q Western blot analysis of YAP levels in 786-O and RCC4 cells treated with DMF in the absence or presence of proteasome inhibitor MG132 (10 μM, 12 h), or lysosome inhibitor NH₄Cl (20 mM, 12 h). R Western blot analysis of YAP ubiquitination levels in 786-O and RCC4 cells transfected with or without Flag-HNF1B. S Western blot analysis of YAP ubiquitination levels in control and HNF1B knockdown 786-O and RCC4 cells. Statistical differences were determined using ordinary one-way ANOVA. ***P < 0.001, ****P < 0.0001.