Fig. 1

YAP/TAZ knockdown impairs TGF-β1-induced Smad3 phosphorylation and Smad Binding Element (SBE) luciferase reporter activity. Primary adult human dermal fibroblasts were transfected with non-specific control siRNA or YAP and/or TAZ siRNAs (400 nM). 48 h after transfection, cells were treated with vehicle (PBS) or TGF-β1 (5 ng/ml) for one hour and then whole cell extracts were prepared for analyses. a Protein levels of phospho-Smad3, total Smad3, YAP, and TAZ were determined by Western blots. Images represent four independent experiments. Full-length blots are presented in Additional file 1: Figure S2. b Quantification of Phospho-Smad3 protein levels. Protein levels were normalized by total Smad3. N = 4, *p < 0.05 vs TGF-β1 control. c Relative mRNA levels of YAP and TAZ in adult human primary dermal fibroblasts. N = 5. d Knockdown levels of YAP and TAZ mRNA by two different sets of YAP and TAZ siRNAs. N = 3, *p < 0.05 vs CTRL siRNA. e Protein levels of total and phospho-Smad3 were determined by capillary electrophoresis immunoassay and normalized to total Smad3. Band intensities were quantified by Compass software. Bands show representative digital images. f YAP/TAZ knockdown inhibits TGF-β1-induced SBE luciferase reporter activity. Fibroblasts were co-transfected with non-specific control or YAP/TAZ siRNAs (400 nM) and Smad Binding Element (SBE) luciferase reporter, and β-galactosidase expression vector. 32 h later, cells were treated with vehicle (PBS) or TGF-β1 (5 ng/ml) for 16 h. SBE luciferase activities were determined 48 h after transfection and normalized to β-galactosidase activity. Bars indicate fold change in SBE reporter activity relative to control siRNA. N = 3, *p < 0.05 vs control siRNA with TGF-β1. g Actin cytoskeleton disassembly inhibits YAP/TAZ nuclear localization. Fibroblasts were treated with vehicle (DMSO) or latrunculin A (Lat-A, 30 nM) for 24 h and then F-actin was stained with phalloidin (red), YAP/TAZ were immunostained (green), and nuclei were stained with DAPI (blue). Scale bars = 25 μm. All images are representative of three independent experiments. h Actin cytoskeleton disassembly inhibits TGF-β1-induced Smad3 phosphorylation. Fibroblasts were treated with DMSO or Lat-A (30 nM) for 24 h, and then cells were treated with PBS or TGF-β1 (5 ng/ml) for one hour. Smad3 phosphorylation (p-Smad3) was determined by immunostaining (red). Nuclei were stained with DAPI (blue). Scale bars = 25 μm. All images are representative of three independent experiments. All data are mean±SEM