Fig. 3.

The effects of Bhlhe40 over-expression on differentiation and metabolism. A Tet-off system was established to stably over-express Bhlhe40 and GFP simultaneously in C2C12 cells (C2C12-Bhlhe40), in which the expression is shut off in the presence of Doxycycline (Dox, 25 ng/ml). The expression of Bhlhe40 mRNA (left panel) and protein (right panel) in myoblasts is shown in (A) and its effects on myogenic differentiation (B) was represented by the fusion index (nuclei number in myotubes/total nuclei number). The effects on mtDNA levels of CMB and MT cells, and on Mitotracker stain intensity, SDH activity, and ROS levels in myotubes are shown in (C), (D), (E), and (F), respectively. (G) Images of C2C12-Bhlhe40 myoblasts transfected with KillerRed-PTS1 expression vector and counterstained with DAPI. The distribution (%) of cells with different numbers of peroxisome/cell and the average peroxisome number/cell are shown in (H) and (I), respectively. * and * *: p < 0.05 and p < 0.01 vs. Dox (+) cells.