Fig 2: Knockdown of StarD7 impaired myogenic differentiation in human primary myoblasts. (a) After transfection with siRNAs against StarD7 (KD) or control siRNA, human primary myoblasts were cultured in differentiation medium for 3 days, then immunostained with anti-MYH6 antibody (green). Nuclei were stained with DAPI (blue). Bars indicate 100 µm. (b) The fusion indexes were calculated, and are presented as the means ± S.D. ***P < 0.001 as compared with control siRNA (Student’s t test). (c) After inducing differentiation, protein levels of MYH4, MYH6, myogenin and StarD7 were analyzed by western blotting. GAPDH was used as a protein loading control. (d) The mRNA levels of myogenin, myomaker, myomerger and PGC-1a were quantified by qPCR after inducing differentiation. Data were normalized to the GAPDH level. Values shown are means ± S.D. from three independent culture dishes. ***P < 0.001 as compared with control siRNA (Student’s t test).

Fig 3: The loss of StarD7 impaired myogenic differentiation in C2C12 cells. (a) After transfection with siRNAs against StarD7 (KD1 or KD2) or control siRNA, C2C12 cells were cultured in differentiation medium for 5 days, then immunostained with anti-MYH6 antibody (green). Nuclei were stained with DAPI (blue). Bars indicate 50 µm. (b) Protein levels of MYH4, MYH6, myogenin and StarD7 were analyzed by western blotting. GAPDH was used as a protein loading control. (c) The mRNA levels of MYH4, MYH6, myogenin, myomaker, myomerger (L), myomerger (S) and PGC-1a were quantified by qPCR. Data were normalized to the GAPDH level. Values shown are means ± S.D. from three independent culture dishes. ***P < 0.001 as compared with control siRNA (one-way ANOVA with Tukey’s post hoc test). (d) Protein levels of StarD7 in WT and StarD7-KO C2C12 cells were analyzed by western blotting. (e) WT or StarD7-KO C2C12 cells were cultured in differentiation medium for 5 days and phase contrast images were obtained.

Fig 4: Reintroduction of StarD7 into the StarD7-KO C2C12 cells restored myogenic differentiation. (a) Cell lysates from WT + EV (empty vector), KO + EV, and KO + StarD7 C2C12 cells were separated by SDS-PAGE and analyzed by western blotting using anti-StarD7 antibody. GAPDH was used as a protein loading control. (b) WT + EV, KO + EV, and KO + StarD7 C2C12 cells were cultured in differentiation medium for 5 days, then immunostained with anti-MYH6 antibody (green). Nuclei were stained with DAPI (blue). Bars indicate 50 µm. (c) The fusion indexes of C2C12 cells were calculated, and are presented as the means ± S.D. ***P < 0.001 as compared with WT + EV cells, and †††P < 0.001 as compared with KO + EV cells (one-way ANOVA with Tukey’s post hoc test). (d) After inducing differentiation, the amounts of MYH4, MYH6, myogenin and myoD protein were analyzed by western blotting. Asterisk indicates a non-specific band. (e) The mRNA levels of MYH4, MYH6, myogenin, myomaker, myomerger (L), myomerger (S) and PGC-1a were quantified by qPCR after inducing differentiation. Data were normalized to the GAPDH level. Values shown are means ± S.D. from three independent culture dishes. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with WT + EV cells, and †P < 0.05, ††P < 0.01, and †††P < 0.001 as compared with KO + EV cells (one-way ANOVA with Tukey’s post hoc test).

Fig 5: Rb1 regulates MSTN and FNDC5 mRNA and protein expression in differentiated C2C12 myoblasts and 3T3-L1 adipocytes. (A-B). The effect of 0, 10, 20, 40 µM Rb1 on MSTN and FNDC5 mRNA expression in differentiated C2C12 myoblasts. (C) Western blot analysis of MYH4, MSTN, and FNDC5 expression in differentiated C2C12 myoblasts. (D-E). Statistical analysis of MSTN and FNDC5 protein levels. (F-G) The effect of 0, 20, and 40 µM Rb1 on MSTN and FNDC5 mRNA expression in differentiated 3T3-L1 adipocytes. (H) Western blot analysis of MSTN and FNDC5 expression in differentiated 3T3-L1 adipocytes. (I-J) Statistical analysis of MSTN and FNDC5 protein levels. (K) The MSTN expression in the culture supernatant of differentiated C2C12 myoblasts. (L) The MSTN expression in the culture supernatant of differentiated 3T3-L1 adipocytes (n = 5). *p < 0.05 vs. CTR group, **p < 0.01 vs. CTR group, # p < 0.05 vs. 10 µM Rb1, ##p < 0.01 vs. 10 µM Rb1, & p < 0.05 vs. 20 µM Rb1, &&p < 0.01 vs. 20 µM Rb1.