Fig. 5

Induction of Smad7 by knockdown of YAP/TAZ is mediated by AP-1. a Nucleotide sequence of the Smad7 proximal promoter. Smad Binding Element (SBE) and Activator Protein 1 (AP-1) binding sites are marked in capital letters. Heavy lines under the sequence denote EMSA probes containing SBE and AP-1 binding elements. Arrow indicates transcription start site. b Primary adult human dermal fibroblasts were co-transfected with non-specific control or YAP/TAZ siRNAs (400 nM), AP-1 reporter construct, and β-galactosidase expression vector. AP-1 reporter activities were determined 48 h after transfection and normalized to β-galactosidase activity. N = 3, *p < 0.05 vs control siRNA. c and d Fibroblasts were co-transfected with non-specific control or YAP/TAZ siRNAs (400 nM). Whole cell extracts were prepared 48 h after transfection. c MMP-1 mRNA levels and (d) MMP-1 protein levels were analyzed by real-time RT-PCR and Western blot, respectively. mRNA levels were normalized to mRNA for housekeeping gene 36B4. Full-length Western blots are presented in Additional file 1: Figure S5A. *p < 0.05 vs control siRNA. e Fibroblasts were co-transfected with non-specific control or YAP/TAZ siRNAs (400 nM), wild-type (− 613 to + 112) or mutant Smad7 promoter/luciferase reporter constructs as depicted in the figure, and β-galactosidase expression vector. Smad7 promoter luciferase activities were determined 48 h after transfection and normalized to β-galactosidase activity. Bars indicate fold change of Smad7 promoter activity relative to control siRNA. N = 3–5, *p < 0.05 vs control siRNA. f and g Fibroblasts were transfected with non-specific control or YAP/ TAZ siRNAs (400 nM) for 48 h. Nuclear extracts were prepared, and DNA/protein complex formation was analyzed by EMSA using AP-1 (f) and SBE (g) probes, as indicated in Fig. 5a (underlined). Closed triangles indicate specific retarded complexes. Open triangle indicates non-specific bands. Arrow indicates Smad3 super-shift band. Results are representative of three experiments. Full-length images are presented in Additional file 1: Figure S5B and S5C. All data are expressed as mean±SEM. h Schematic of cross-talk between YAP/TAZ and TGF-β/Smad pathways involving AP-1 regulation of Smad7