Figure 8.

Mtf1 phenotype is restored by MTF1 WT but partially recovered by the MBS mutant. A) Representative Western blot of primary myoblasts and myoblasts transduced with either scr shRNA or the shRNAs against Mtf1-1. The Mtf1 shRNA-1 myoblasts were transduced with the MTF1 WT and MBS mutant. Samples were obtained for proliferation and 24 h after inducing differentiation. MTF1 levels were detected with the MTF1 antibody and the exogenous protein was detected with an anti-Flag antibody. SERCA levels were monitored as a differentiation marker. PI3K was used as loading control. B) Representative confocal microscopy images of differentiating primary myoblasts at 24 using an anti-Flag antibody to detect the localization of endogenous MTF1 (red) and DAPI (blue). C) Representative light micrographs of differentiating myoblasts immunostained for myogenin at 24 h. Images depicted in A–C are representative of ≥3 independent biologic experiments. D) Calculated fusion index for Mtf1-shRNA–transduced myoblasts and recovered with MTF1 WT or the MBS mutant; values for WT, and shRNA-1 MTF1 corresponds to the values shown in Fig. 2E. E) ChIP-qPCR for MTF1 binding to the Myogenin promoter in the myoblasts described in (A) differentiated with insulin or Cu. F) Whole cell Cu content of proliferating and differentiating primary myoblasts determined by AAS. G, H) Nuclear (G) and cytosolic (H) Cu content of proliferating and differentiating primary myoblasts. Metal determination was performed by AAS. Box plots represent the distribution of the data obtained from 3 independent biologic experiments ± sd. *P < 0.05, **P < 0.01, ***P < 0.001, ****P ≤ 0.0001.