Fig. 3

Knockdown of YAP/TAZ or inhibition of YAP/TAZ nuclear localization induces inhibitory Smad7. Primary adult dermal fibroblasts were transfected with non-specific control or YAP/TAZ siRNAs (400 nM). Whole cell extracts were prepared 48 h after transfection. a Smad7 mRNA levels. mRNA levels were quantified by real-time RT-PCR. mRNA levels were normalized to mRNA for housekeeping gene 36B4. N = 5, *p < 0.05 vs CTRL siRNA. b Smad7 protein levels. Protein levels were determined by Western blot and normalized by β-actin (loading control). N = 3, *p < 0.05 vs CTRL siRNA. Full-length blots are presented in Additional file 1: Figure S4. c TGF-β pathway components mRNA expression. N = 3–6. d Actin cytoskeleton disassembly induces Smad7 protein. Primary adult human dermal fibroblasts were treated with vehicle (DMSO) or latrunculin A (Lat = A, 30 nM) for 24 h, and then cells were immunostained for Smad7 (top panel, green), YAP/TAZ (middle panel, green), and phalloidin (lower panel, red). Nuclei were stained with DAPI (blue). Scale bars = 25 μm. The staining intensities and cell surface areas were quantified by ImageJ software. *p < 0.05 vs DMSO. All images are representative of three independent experiments. e Actin cytoskeleton disassembly induces Smad7 mRNA. mRNA levels were quantified by real-time RT-PCR and normalized to mRNA for housekeeping gene 36B4. N = 3, *p < 0.05 vs DMSO. f Limiting cell spreading induces Smad7 protein. Fibroblasts were plated on standard tissue culture plates (left panels) or poly-L-lysine (PLL) coated plates (right panels). 24 h later, cells were immunostained for Smad7 (top panel, green), YAP/TAZ (middle panel, green), and phalloidin (low panel, red). Nuclei were stained with DAPI (blue). Scale bars = 25 μm. The staining intensities and cell surface areas were quantified by ImageJ software. *p < 0.05 vs standard tissue culture plate. All images are representative of three independent experiments. g Limiting cell spreading induces Smad7 mRNA. mRNA levels were quantified by real-time RT-PCR and normalized to mRNA for housekeeping gene 36B4. N = 3, *p < 0.05 vs DMSO. All data are mean±SEM