FIGURE 5.

Deletion of TAK1 inhibits MyoD-induced differentiation in fibroblasts. TAK1^+/+ and TAK1^−/− MEF were transduced with Ad.MyoD for 24 h at multiplicity of infection 50. The cells were then incubated in DM for different time intervals. A, myotube formation was measured after 48 h of incubation in DM by performing immunofluorescence using MF20 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. The top photomicrographs (green fluorescence protein) show equal transduction of TAK1^+/+ and TAK1^−/− MEF by Ad.MyoD vector. The bottom photomicrographs show that myotube formation (red color) is significantly reduced in TAK1^−/− MEF cultures compared with TAK1^+/+. B, Western blot showed that the levels of MyHCf were reduced in TAK1^−/− MEF compared with TAK1^+/+ after incubation in DM. Immunoblots also show equal levels of MyoD protein in both TAK1^+/+ and TAK1^−/− MEF and the presence of truncated TAK1 protein in TAK1^−/− MEF. Wt, wild type. C, levels of CK were also found to be significantly reduced in TAK1^−/− MEF compared with TAK1^+/+ MEF after 24 and 48 h of incubation in DM. *, p < 0.01, values significantly different from TAK1^+/+ MEF at corresponding time point. D, TAK1^+/+ and TAK1^−/− MEFs were transfected with pcDNA3-MyoD plasmid along with pSK-Luc plasmid in a 1:10 ratio for 24 h. Cells were then incubated in DM for 48 h, and the activation of skeletal α-actin promoter was monitored by measuring luciferase activity. Data presented here show a significant reduction in the activation of skeletal α actin promoter in TAK1^−/− MEF compared with TAK1^+/+ MEF. #, p < 0.01, value significantly different from TAK1^−/− MEF.