Fig. 4: Excess PrPC strongly inhibits skeletal muscle cell autophagy and blocks myoblast differentiation.
Immunofluorescence imaging of C2C12 mouse myoblasts (control), C2C12 myoblasts stably overexpressing WT PrPC, and C2C12 myoblasts KO for PrPC cultured until their confluence reached 90% and then incubated with differentiation medium for 3 (a) and 5 (b) days, respectively, using antibodies against PrPC (red) and LC3B (green) and staining with DAPI (blue). The enlarged regions in the lower left corner (a) or the lower right corner (b) of the merged images show 4-fold enlarged images from the same images. Scale bars, 7.5 (a) and 50 (b) μm, respectively. We have replaced the first row in (a) with a correct version of control, in which LC3B did not overlap with PrPC in most fields of view. c Western blot for PrPC and the autophagy markers ATG5 and LC3B during the above three cell lines incubated with differentiation medium for 3 and 5 days, respectively. β-actin served as the protein loading control. The relative amount of ATG5 (d) or LC3B-II (e) in the above cell lines (control, orange; WT PrPC, blue; and KO for PrPC, gray) (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in n = 3 independent experiments. One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05. Source data are provided as a Source Data file.
