Fig. 3

YAP1 activates actin and cytoskeletal proteins to transform NFs to CAFs by regulating SRC. a The association of YAP1 and SRC in prostate cancer was analysed online at http://gepia.cancer-pku.cn/. Pearson R = 0.32. b-c The mRNA expression levels of YAP1, α-SMA and SRC in the indicated four cell lines were detected by qRT-PCR. d The protein expression levels of YAP1, α-SMA, SRC and p-SRC in the indicated four cell lines were detected by western blot. GAPDH was used as an endogenous reference gene. e Western blot was used to detect the protein expression levels of TEAD1, YAP1, p-YAP1, SRC, p-SRC and α-SMA after siTEAD1 transfection of the CAFs. GAPDH was used as an endogenous reference gene. f Western blot was used to detect the protein expression levels of TEAD1, YAP1, p-YAP1, SRC, p-SRC and α-SMA when they were knocked down or overexpressed in CAFs. GAPDH was used as an endogenous reference gene. g The interaction between YAP1 and TEAD1 in the CAFs was determined by the co-IP assay. The relative levels of YAP1 or TEAD1 in these cells were determined by western blot using a YAP1 or TEAD1 antibody. h Chromatin immunoprecipitation (ChIP) of the CAFs was performed with control IgG and TEAD1 antibodies. The precipitation of the SRC promoter was examined by PCR. i A dual-luciferase reporter assay driven by the SRC promoter was co-transfected in the presence or absence of YAP1 or TEAD1. The relative luciferase activities were determined by calculating the ratio of firefly luciferase activities over Renilla luciferase activities. Three independent experiments were conducted, with the means±s.d. of the relative luciferase activities shown. j Western blot was used to detect the protein expression levels of SRC, YAP1, TEAD1, MYL9, F-actin, paxillin and α-SMA after siSRC transfection of the CAFs. GAPDH was used as an endogenous reference gene. k qRT-PCR detection of mRNA expression levels of MYL9, F-actin and paxillin in the CAFshYAP1 group. l Western blot was used to detect the protein expression levels of SRC, MYL9, F-actin and paxillin in the CAFshYAP1 group. GAPDH was used as an endogenous reference gene