Fig. 3. TGFβ signaling limits cell fusion.

a Experimental scheme. Primary myoblasts seeded at low density (5000 cells/cm²) were differentiated for 2 days, split, and re-plated at high density (75,000 cells/cm²) and cultured for 2 more days. b Immunofluorescent staining for MYOGENIN of primary myocytes pre-differentiated for 48 h and re-plated at high density confirms that >90% of cells express Myogenin. N = 7 biologically independent experiments. c qRT-PCR analysis for Myogenin and Smad7 transcript expression of re-plated primary myocytes cultured for 24 h with or without TGFβ1 recombinant protein. Although TGFβ1 stimulation activates Smad7 expression, it does not affect Myogenin transcript levels. N = 6 primary cell cultures. d Immunofluorescent staining for the MYOSIN HEAVY-CHAIN isoforms (Pan-MyHC) of re-plated primary myocytes cultured for 48 h. e Percentage of Pan-MyHC-expressing cells of re-plated myotubes shows that cells were differentiated in all conditions. N = 5 biologically independent experiments. f Fusion index of re-plated myotubes reveals that TGFβ stimulation inhibits fusion. N = 5 biologically independent experiments. g Percentage of nuclei in the smallest and largest myotube classes. TGFβ-treated myotubes are characterized by less nuclei per myotube. N = 11 (control) and 15 (TGFβ1) biologically independent experiments. Scale bars: b 400 μm; d 200 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. ** and *** denote a significant difference with the Control group of P < 0.01 and P < 0.001, respectively. NS not significant. Source data are provided as a Source Data file.