Fig. 4. SDF1 attenuates myogenic differentiation of C2C12 cells and primary myoblasts. (A,C) At the switch from growth to differentiation conditions, C2C12 cells were treated with the indicated concentrations of SDF1 for 24 hours or 48 hours and then analyzed for (A) MyoD or (C) myogenin expression, respectively, by western blotting. Numbers represent average MyoD or myogenin protein levels (arbitrary units), corrected for β-actin levels, as determined by densitometric analysis of blots from five independent experiments. SDF1 significantly attenuates myogenic differentiation of C2C12 cells, maintained under differentiation conditions (MyoD, F=7.9, dF=15, P<0.005; myogenin, F=8.6, dF=15, P<0.005, ANOVA). (B,D) Real time (RT)-PCR analysis of C2C12 cells, maintained for 24 hours under differentiation conditions and the additional presence of SDF1 at different concentrations, for (B) MyoD and (D) myogenin mRNA levels. Data represent the mean ±s.d. from four independent experiments. Note that SDF1-induces a significant loss in myogenin mRNA levels (F=13.7, dF=12, P<0.001; ANOVA) but does not affect MyoD mRNA levels. (E) C2C12 cells were treated at the switch from growth to differentiation conditions with SDF1 (10 ng/ml) and analyzed for MHC expression after 2 days and 5 days by western blotting using the MF20 antibody. Staining for β-actin served as a loading control. MHC was undetectable in C2C12 cells maintained with SDF1 for 5 days. (F) Cultures of primary myoblasts and myogenic precursors were established from rodent hindlimb musculature as described in Materials and Methods. Cells were maintained for 4 days in the presence or absence of SDF1 (10 ng/ml) and subsequently analyzed for myogenin expression levels by western blotting. As found for C2C12 cells, SDF1 inhibits differentiation of primary myoblasts.