Figure 5.

Insulin and lithium chloride cooperate to promote hypertrophy in differentiating C2.7 myoblasts. (A) C2.7 cells were cultured in a proliferation medium for 3 d and then transferred to differentiation medium for 48 h. At that time, differentiating myoblasts were stimulated or not (control 3%) with insulin at 3 μg ml^-1 (insulin), lithium chloride at 10 mM (LiCl), or both of them (insulin+LiCl). Twenty-four hours after stimulation (after a total of 72 h of differentiation), cells were fixed and photographed by phase contrast. Bar, 150 μm. (B) For size measurements and nuclei quantification, cells were fixed with formalin for 7 min and stained with bisbenzimide 33258. The mean diameter of the largest myotubes was evaluated on the basis of the 10 largest myotubes in six different randomly chosen microscopic fields per condition and averaged from four different experiments. To quantify the increase in the fusion process, the cultures also were characterized by counting the maximum number of nuclei present in the 10 largest myotubes as described for the size measurements.