Fig. 7

LC3B-II expression is increased but autophagic flux is decreased in differentiating dystrophin-deficient myoblasts. Myoblasts were cultured in GM until 80–90% confluent and culturing in DM for 10 days prior to total protein extraction and immunoblot analysis with antibodies recognizing LC3B with α-tubulin expression as a loading control. Densitometry analysis of b LC3B-I expression, c LC3B-II expression, and d the ratio of LC3B-II/LC3B-I. For flow cytometry analysis, three different conditions were set up for each stage: (1) unstained cells (negative control); (2) stained cells; and (3) stained cells with chloroquine treatment (positive control). Chloroquine treatment consisted of 4 h’ incubation at 37 °C. Cells were trypsinized and incubated with Cyto-ID Green stain solution before analysis using the FITC channel of a CyAn B flow cytometer (Beckman Coulter, USA). Data analysis was performed using FCS Express 6 Plus Research Edition De Novo Software, USA). The density plot images represent non-differentiated e C2C12 myoblasts, f dfd13 myoblasts; day10 differentiation g C2C12 myoblasts and h dfd13 myoblasts. The histogram overlays i–l represent each neighboring dot plot. m The percentage of autophagosomes detected in myoblasts and n percentage of autophagic flux within myoblasts. All bar graphs represent an average of three repeats from different samples. All data are presented as mean ± S.D. Significantly different: * (p < 0.05) and ** (p < 0.01) compared to C2C12 myoblasts. # (p < 0.05) when compared with ND; § (p < 0.05) when compared to DM + CLQ