Fig. 3

miR-9-5p attenuates Smad-dependent TGF-β signaling. a Western blot analysis (left) and quantification (right) of pSmad2 protein levels in HDF cells transfected with 40-nM pre-miR-9-5p or pre-miR-NC and treated with TGF-β1 (5 ng/ml) at the indicated times (a.u., arbitrary units). The bar graph shows values after correction by Smad2 expression and normalized to control conditions. b Immunofluorescence staining of Smad2/3 (green) in HDFs treated with TGF-β1 (5 ng/ml) for 30 min after transfection with 40-nM pre-miR-9-5p or pre-miR-NC. Nuclei were stained with DAPI (blue), and F-actin was stained with phalloidin (red). Scale bars: 100 μm. c Western blot analysis after nuclear/cytoplasmic fractionation (left) and quantification (right) of Smad2/3 protein levels in HDF cells transfected with 40-nM pre-miR-9-5p or pre-miR-NC and treated with TGF-β1 (5 ng/ml) at the indicated times (a.u., arbitrary units). The bar graph shows values after correction by GAPDH (cytosol) and LMNA (nucleus) expression and normalized to control conditions. All bar graphs represent mean ± SEM of three independent experiments, **P < 0.01 compared to control cells and ^# P < 0.05 compared to its corresponding negative control time point