FIGURE 3.

Characterization of reserve cells and endogenous expression of Capn3. A, schematic representation of protocol used to study the different subpopulations of C2C12 cells. C2C12 myoblasts, transfected or not, were maintained in proliferation for 24 h. Differentiation was then induced over 4 days, and the populations of myotubes and reserve cells were separated, as described under “Experimental Procedures.” B, 40 μg of total cell extract of each subpopulation (myoblasts, reserve cells, and myotubes), just as reserve cells that had been put back in proliferative medium for 12 h (reserve cells + 12 h), were analyzed by SDS-PAGE and immunoblotting with antibodies specific for MyoD, Pax7, myogenin, and β-tubulin. C and D, the relative endogenous Capn3 and myogenin expression levels were quantified in the three populations by qRT-PCR, as described under “Experimental Procedures.” The relative expression level of Capn3 or myogenin is presented such that 1 indicates the expression level in myoblasts. The data are presented as mean ± S.D. (n = 3). The single asterisks indicate data that are significantly different according to an ANOVA test for p < 0.05.