FIGURE 6.

Effect of calpain 3 on the MRFs. A, C3H10T1/2 cells were transfected with a plasmid encoding one of the four MRFs (pEMSV-Myf5, pEMSV-MRF4, pEMSV-myogenin, or pEMSV-MyoD) by itself or along with pEMSV-CAPN3. Cells were maintained in proliferation medium for 24 h before being harvested. Total cell extract was prepared, and 40 μg was analyzed by SDS-PAGE and immunoblotting with antibodies specific for MyoD, Myf5, MRF4, myogenin, and β-tubulin. B–E, the transcriptional activity of each MRF was measured by analysis of MCK promoter-driven expression of luciferase. At 48 h after transfection, the C3H10T1/2 cells were harvested, and transactivation of the reporter gene was determined by a luciferase assay, as described under “Experimental Procedures.” Luciferase activity is expressed such that 100% indicates the maximum activity measured for each MRF tested without calpain 3. F and G, the transcriptional activity of MyoD was measured by use of a skeletal muscle reporter gene (CAT) under the control of a minimal promoter consisting of four repeats of the E-box element. At 48 h after transfection, the C3H10T1/2 cells were harvested, and transactivation of the reporter gene was determined by a CAT-ELISA assay, as described under “Experimental Procedures.” The amount of CAT is expressed such that 100% indicates the maximum value measured for MyoD without calpain 3. The MRFs and calpain 3 expression plasmids that were transiently transfected into the fibroblast cells (C3H10T1/2) are indicated below the graphs, and +, ++, and +++ indicate the relative amount of plasmid DNA used. The data represent mean ± S.D. (n = 3). The asterisks indicate data that are significantly different according to an ANOVA test for p < 0.05.