Fig. 4

YAP and AP-1 are involved in ET-1/ILK-induced transcriptional activation of ZEB1. A Schematic representation of ZEB1 promoter reporter construct carrying the binding motifs of AP-1 and TEAD4 obtained from JASPAR. B Total extracts of PMOV10 cells transfected with SCR, si-ZEB1, si-YAP, or si-JUN for 72 h and stimulated with ET-1 and/or treated with macitentan for 48 h were IB with anti-ZEB1 Ab. β-actin was used as loading control. C Total extracts of PMOV10 cells stimulated with ET-1 and/or treated with macitentan for 30 min as indicated were IB with anti-ILK Ab. β-actin was used as loading control. D Nuclear extracts of PMOV10 cells transfected with SCR or si-ILK for 72 h and stimulated with ET-1 for 6 h were IB for ZEB1 or YAP. PCNA was used loading control. E ZEB1 gene expression in PMOV10 cells transfected for 72 h as in B or with si-ILK and stimulated as indicated for 24 h was analyzed by q-RT-PCR and normalized to cyclophilin-A. Values are the mean ± SD expressed as fold over control (n = 3; ***p < 0.001; #: p value was calculated vs. SCR + ET-1). F ZEB1 promoter activity was analyzed in PMOV10 cells stimulated for 24 h as indicated and transfected with SCR, si-ZEB1, si-YAP, si-JUN, or si-ILK for 72 h and together with a reporter plasmid for ZEB1 promoter for 48 h. Values are the means ± SD expressed as fold over control (n = 3; *p < 0.05; **p < 0.01; #: p value was calculated vs. SCR + ET-1)