Fig. 1

ActRII and BMP ligands promote distinct SMAD signaling yet similar differentiation blockade in human myoblasts​. (A) Western blotting analysis for SMAD signaling with BMP ligands at 300 ng/ml (upper left), SMAD signaling with a dose response of ActRII and BMP ligands (bottom left), and non-SMAD signaling with a dose response of ActRII and BMP ligands (right). Human transduced myoblasts (iHUSKMDC) were acutely stimulated with vehicle as a negative control, BMP ligands, or ActRII ligands for 30 min. (B) Western blotting analysis with combined ligands. Human iHUSKMDC myoblasts were stimulated with vehicle as a negative control or with increasing doses of ActA ± BMP9 (left) or ActA ± BMP7 (right) at 1, 10, or 100 ng/ml. (C) Human myoblast differentiation assay with individual ligands. iHUSKMDC myoblasts were differentiated into myotubes with vehicle as negative control, TNFα (30 ng/ml) or IGF1 (10 ng/ml) as positive controls or increasing doses of ActRII or BMP ligands (0.3, 30, or 300 ng/ml). Representative images are shown per treatment, with myotubes (green) identified using anti-MyHC antibody staining and nuclei (blue) using DAPI staining. ​Scale bar, 100 μm. Differentiation was quantified by evaluating the percentage of nuclei within myotubes that were positively identified using anti-MyHC antibody staining. Differences between groups were analyzed using one-way ANOVA (compared to UNT group). Data are means ± SD. Asterisks indicate differences with the control. Statistical analyses were conducted with a One-way ANOVA. *p ≤.05; **p ≤.01; ***p ≤.001; ****p ≤.0001 compared to control