FIGURE 3.

ASK1 promotes myoblast differentiation. A, lysates of C2C12 cells that stably express shAsk1-1, shAsk1-2, or control (pSuper) vectors were immunoblotted with antibodies to ASK1 and MHC and to cadherin as a loading control. B, photomicrographs of C2C12 cells shown in A that were cultured in DM for 3 days, fixed, and stained with antibody to MHC. C, quantification of myotube formation by the cell lines shown in B. Values represent the means of triplicate determinations ± 1 S.D. The experiment was repeated three times with similar results. Asterisks indicate difference from the control at p < 0.01. D, lysates of cell lines shown in B were immunoblotted with antibodies to MHC, myogenin, or troponin T and to cadherin as a loading control. E, lysates of C2C12/control, C2C12/ASK1, or C2C12/ASK1(KN) cells from proliferating high density cultures were immunoblotted with antibodies to ASK1 and to cadherin as a loading control F, C2C12 cells were stably transfected with expression vectors for ASK1, a kinase-negative form of ASK1 (ASK1(KN)), or the control. Cells at differentiation day 2 were immunostained for MHC (red), and nuclei were visualized by DAPI staining (blue). G, quantification of myotube formation shown in F. Values represent the means of triplicate determinations ± 1 S.D. The experiment was repeated three times with similar results. The asterisk indicates difference from the control at p < 0.01. H, lysates of C2C12/pcDNA, C2C12/ASK1, or C2C12/ASK1(KN) cells cultured in DM for 2 days were immunoblotted with antibodies to MHC and to cadherin as a loading control.