Figure 6: Effects of WCUP on the differentiation of C2C12 myoblasts and on the wasting of C2C12 myotubes.

(A) C2C12 myoblasts at a density of 80% were differentiated by incubating in DM or in WCUP-treated or -untreated CT-26 CM after a 1:5 dilution in DM. After 4 days, cells were observed under an inverted microscope (upper) and detected for MyH expression by ICC (lower). Myotube length (8–10 tubes per group) and myotube number (5 fields per group) were examined and data are expressed as means ± SD. Statistical significance was evaluated with Student t-test. ^#p < 0.001 vs. DM, **p < 0.01 and ***p < 0.001 vs. WCUP-untreated CT-26 CM. (B) The levels of MyH in cells were measured by Western blotting, and the relative band intensities were calculated after normalization to tubulin expression. (C) The C2C12 myotubes were incubated in DM or in WCUP-treated or -untreated CT-26 CM after a 1:5 dilution in DM. After 2 days, myotube degradation was observed under an inverted microscope (upper), and MyH expression was detected by ICC (upper). Myotube length (8–10 tubes per group) and myotube number (5 fields per group) were calculated and data are expressed as means ± SD. Statistical significance was evaluated with Student t-test. ^#p < 0.001 vs. DM, ***p < 0.001 vs. WCUP-untreated CT-26 CM. (D) The levels of MyH, p65 phosphorylation, and Akt phosphorylation were measured by Western blotting after incubating C2C12 myotubes in DM or in WCUP-treated or -untreated CT-26 CM for 48 h. Relative band intensities were calculated after normalization to tubulin expression. (E) WCUP-treated or -untreated CT-26 CM were concentrated using a Centricon (Amicon Ultra centrifugal filter, 30 K; Millipore Co., Billerica, MA, USA) and then analyzed for Mstn expression by Western blotting. The full size gels and blots were shown in the Supplementary Fig. S10 and band of interest is indicated with an arrow.