Fig. 2

BMP signaling is selectively perturbed in ∆Ig3-MuSK myogenic cells. A and B MuSK expression: A Unpermeabilized WT and ∆Ig3-MuSK myoblasts cells were fixed, and cell surface MuSK was visualized by immunostaining with an antibody directed against the MuSK Ig2 domain (red). Note that comparable levels and distribution of immunostaining were observed in both genotypes. B WT and ∆Ig3-MuSK myoblast cell lines express comparable levels of MuSK transcript as assessed by qRT-PCR. Data are means ± SEM from five biological replicates and two independent experiments. C–G BMP signaling: C WT and ∆Ig3-MuSK myoblasts were treated with 20 ng/ml BMP4 for 15 min and immunostained for pSmad1/5. Note the increased intensity of pSmad staining in nuclei of BMP4-treated WT compared to ∆Ig3-MuSK cells. Data are from three independent experiments with three biological replicates per group. D pSmad1/5 and total Smad1 in BMP4-treated cells (as in C) were assessed by Western blotting. E The level of BMP4-induced pSmad1/5, normalized to total protein, was reduced ∆Ig3-MuSK as compared to WT cells. Data are means ± SEM of three biological replicates and independent Western blots (****p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons). See also Supplementary Fig. S2 for protein staining of these blots. F Cells were treated with 25 ng/ml BMP4 for 2 h, and Car3 transcript levels were assessed. Data are mean ± SEM of 5–6 biological replicates per condition and replicated twice (****p < 0.0001, ***p < 0.001, *p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons). G Cultured primary myotubes from WT and ∆Ig3-MuSK mice were treated with 25 ng/ml BMP4 for 2 h, and levels of Wnt11 mRNA were measured by qPCR. Note that ∆Ig3-MuSK myotubes show reduced Wnt11 expression in response to BMP4 stimulation. Data are means ± SEM from 6 biological replicates from two experiments (****p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons)