Fig 2.

Map4k4 silencing promotes myotube formation in C2C12 cells. C2C12 myoblasts were transfected with scrambled siRNA or siRNA against Map4k4. Twenty-four hours later, the cells were transferred to DM for the indicated times. (A) (Top) Efficiency of Map4k4 knockdown as determined by immunoblotting. (Bottom) Cells were fixed and immunostained for MyHC, and myoblast differentiation was observed by fluorescence microscopy (green, MyHC; blue, DAPI). Magnification, ×100. Data are representative of at least three independent experiments. (B) Fractions of myotubes with the indicated numbers of nuclei were quantified among 100 randomly chosen myotubes after 72 h in DM. (C) The fusion index for day 3 myotubes was calculated from the ratio of the number of nuclei in MyHC-positive myotubes to the total number of nuclei in one field for five random microscopic fields. (D) Myotube diameters were measured with day 3 myotubes. Results represent means and SEM for three independent experiments. ***, P < 0.001.