Figure 3.

A combination of five miRNAs accelerates differentiation of human myoblasts and improves insulin sensitivity downstream of FAK signaling. Human primary myoblasts were transfected with equal concentrations of control antagomirs or antagomirs against the indicated miRNAs, either as single antagomirs or in combination (Ant-5x). Twenty-four hours after transfection, differentiation was induced for two days (A, B, C, E, G) or up to five days (D, F). A. Gene expression of myogenic regulatory factors and eMHC was analyzed by qRT-PCR and normalized for 18S RNA, n = 5. B. Protein expression of myogenin and eMHC was analyzed by western blot and normalized to GAPDH (n = 4–6). C. Luciferase vectors harboring either the myogenin 3′UTR (n = 4) or promoter region (n = 5) were transfected with miRNA mimics or antagomirs respectively. Myogenin mRNA from control and Ant-5x treated samples was measured by qRT-PCR at the indicated time points following Actinomycin D administration (n = 3). D. Time course of myogenin and eMHC protein expression during five days of differentiation, normalized to GAPDH (n = 4–6). E. Immunofluorescent analysis of myotube formation using anti-desmin and wheat germ agglutinin (WGA). Fusion index was calculated as percentage of nuclei present in cells containing at least two nuclei compared to all nuclei per well (scale bar 100um, n = 4). F. Phosphorylation of p38 MAPK, AKT and FAK during the first three days of differentiation, n = 4. G. Insulin-dependent glycogen synthesis, n = 3. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, A, B, C: One way-ANOVA with Dunnett's multiple comparison test, D,E, F: student's t test. G: Two way-ANOVA. All results are shown as mean ± SEM.