Fig. 2

R-SMAD overexpression blocks human myoblast differentiation and ActRII inhibition mitigates ligand inhibitory effects. (A) Differentiation assay and western blotting analysis validation for SMAD2 and SMAD3 over-expressing lines. Stable skeletal muscle cell lines (iHUSKMDC) with SMAD2 or SMAD3 over-expression (OE) were differentiated into myotubes. Representative images (n = 3 replicates) are shown for each treatment, using anti-MyHC antibody staining for myotubes (green) and nuclei (blue) using DAPI staining. Scale bar, 100 μm. Western blotting analysis at day 0 of differentiation was used to confirm overexpression and signaling with SMAD-selective antibodies. (B) Differentiation assay and western blotting analysis validation for SMAD1 and SMAD5 over-expressing lines. Stable skeletal muscle cell lines (iHUSKMDC) with SMAD1 or SMAD5 over-expression (OE) at lower and higher levels were differentiated into myotubes. Representative images (n = 3 replicates) are shown for each treatment, using anti-MyHC antibody staining for myotubes (green) and nuclei (blue) using DAPI staining. Scale bar, 100 μm. Western blotting analysis at day 0 of differentiation was used to confirm relative overexpression and signaling with SMAD-selective antibodies across the lines. (C) Differentiation assay and western blotting analysis to assess ActRII inhibition effects. Human myoblasts (iHUSKMDC) were pre-treated with a selective anti-ActRIIA/B neutralizing antibody or isotype control (25 or 100 nM) followed by stimulation with 10 ng/ml ActA, BMP7, or BMP9. Scale bar, 100 μm. Representative images (n = 2) are shown per treatment, with myotubes (green) identified using anti-MyHC antibody staining and nuclei (blue) using DAPI staining. ​Scale bar, 100 μm