Fig. 8. PKC mediates the inhibitory influences of SDF1 on myogenic differentiation. (A) C2C12 cells were maintained for 48 hours with either myristoylated PKC peptide inhibitor (2 µM) alone or in combination with the indicated concentrations of SDF1, and subsequently analyzed for myogenin expression by western blotting. Protein loading was controlled by GAPDH staining. Myogenin levels increased with myristoylated PKC peptide inhibitor and remained unaffected by SDF1. Data are given in Table 2. (B) C2C12 cells were transfected overnight with PKC siRNA or non-homologous (nh) siRNA, and maintained further in DMEM containing 1% HS and SDF1 (10 ng/ml). After 48 hours, cells were lysed and myogenin expression was determined by western blotting. Immunoreactive protein bands were measured by densitometry and corrected for GAPDH. Numbers represent average protein level ± s.d. (arbitrary units) as determined in three independent experiments. Myogenin protein levels increased in C2C12 cells transfected with PKC siRNA. This increase was not affected by the additional presence of SDF1. By contrast, C2C12 cells transfected with nh siRNA showed unchanged expression levels of myogenin which declined in the presence of SDF1. ^aP<0.05, PKC siRNA vs control; ^bP<0.05, PKC siRNA+SDF1 vs control. (C) Assessment of PKC expression in cells transfected with PKC siRNA. C2C12 cells were transfected with PKC siRNA or non-homologous (nh) siRNA. Levels of PKC were determined by western blotting. Numbers indicate average levels of PKC ± s.d. (arbitrary units) as determined in five independent experiments. Note that PKC levels significantly decline (P<0.001) in C2C12 cells transfected with PKC siRNA as compared with cells transfected with nh siRNA.