Figure 3. The overexpression of Stabilin-2 enhances myotube formation in myoblasts.

(a) C2C12/Stab2 and C2C12/Mock cells were induced to differentiate for the indicated times. Cells were then fixed and immunostained with anti-MyHC antibody. Representative microscopic fields are shown. Scale bar, 100 μm. Red boxes are shown at higher magnification. (b) C2C12/Stab2 and C2C12/Mock cells were induced to differentiate for the indicated times, and fusion indices were calculated. Data are presented as mean±s.d. of three independent experiments. (c) After 5 days of differentiation (DM5), the percentage of nuclei present in MyHC-positive myotubes with the indicated number of nuclei were quantified in C2C12/Stab2 and C2C12/Mock cells. Data are presented as mean±s.d. of three independent experiments. (d,e) C2C12/Stab2 and C2C12/Mock cells were induced to differentiate for the indicated times. The levels of embryonic MyHC (Myf3, d) and myogenin (MyoG, e) mRNA were analysed by quantitative real-time PCR. Data are presented as mean±s.d. of three independent experiments. (f) The levels of MyHC and myogenin proteins were analysed in C2C12/Stab2 and C2C12/Mock cells during differentiation by immunoblotting. Full-size blots are shown in Supplementary Fig. 15. (g,h) L cells stably transfected with stabilin-2 expression vector (L/Stab2) or empty vector (L/Mock) were incubated in growth medium (GM) or fusion medium (FM). Representative images (g) are shown. Scale bars, 100 μm. Fusion indices of stabilin-2-expressing cells at the indicated time points are shown in graph (h). Data are presented as mean±s.d. of at least three independent experiments. (i) Differential interference contrast (DIC) images from Supplementary Movie 1 showed cell–cell fusion in stabilin-2-expressing cells. Asterisks indicate statistical significance (*P<0.05, **P<0.01, ***P<0.001, Student's t-test).