Fig. 2: RNF214 augments Hippo-regulated transcription.

a mRNA analysis of TEADs target genes in RNF214-knockdown Hep3b cells. An siRNA-resistant cDNA of RNF214 resumed the mRNA levels of three target genes in RNF214-silenced cells. b mRNA analysis of TEADs target genes in Huh7 cells with RNF214 knockdown. c Serum induces ANKRD1, CTGF and CYR61 transcription. HEK293A cells were transfected with siRNF214, starved in serum-free medium for 12 h and then stimulated with 10% serum for the indicated time. d Rnf214^−/− MEFs show low activity of serum induced-TEAD transcription. MEF cells were starved in serum-free medium for 12 h and then stimulated with 10% serum. “*” indicates non-specific band. e CTGF-luciferase reporter assay. HEK293T cells were co-transfected with the reporter system along with the increasing amounts of Flag-RNF214 (0, 100, 200, or 400 ng). f Gal4-TEAD4/9xUAS-luciferase reporter assay. The transcriptional activities of YAP-TEAD4 were measured based on YAP’s ability to co-activate the Gal4 DNA binding domain fused to TEAD4 (Gal4-TEAD4) on the 9xUAS-luciferase reporter. Increasing amounts of Flag-RNF214 (0, 100, 200 or 400 ng) were co-transfected into the HEK293T cells with the reporter system. g Schematic diagram of RNF214 domains. h, i RNF214 enhances TEADs transcriptional activities depending on its ubiquitin ligase activity. h HEK293T cells were transfected with Flag-RNF214 wild type (WT) or RING finger deletion mutant (RD) along with Gal4-TEAD4/9xUAS-luciferase. Western blotting was employed to verify expression consistency between WT and RD RNF214. i An siRNA-resistant cDNA of RNF214 RD mutant was stably introduced into Huh7 cells and then endogenous RNF214 was knocked down. j, k The coiled-coil domain of RNF214 is essential for its effect. j HEK293T cells were transfected with Flag-RNF214 WT or the coiled-coil deletion mutant (CCD) along with Gal4-TEAD4/9xUAS-luciferase. k An siRNA-resistant cDNA of RNF214 CCD mutant was stably delivered into Huh7 cells and then endogenous RNF214 was knocked down. Data are presented as mean ± SD. P values were calculated using two-sided unpaired Student’s t test; n = 3 biologically independent samples in experiments (a, b, e, f, h–k). Experiments in figures (c, d) were repeated twice. Source data are provided as a Source Data file.