Figure 5: Effects of WCUP on C2C12 myoblast proliferation.

(A) C2C12 myoblasts were treated with increasing concentrations of WCUP (50-1000 μg/mL) diluted in GM for 48 h, and then cell viability was determined using a CCK-8 kit. The data are representative of independent experiments performed in triplicate and expressed as means ± SD. (B) C2C12 myoblasts were incubated with WCUP-treated or -untreated CT-26 CM after 1:3 and 1:5 dilutions in GM. After 36 h of incubation, viable cells were measured using a CCK-8 kit. The data are means ± SD and representative of independent experiments performed in triplicate. Statistical significance was evaluated with Student t-test. ^#p < 0.001 vs. GM, **p < 0.01 and ***p < 0.001 vs. WCUP-untreated CT-26 CM. (C) After exposure to WCUP-treated or -untreated CT-26 CM for 36 h, cells were observed under an inverted microscope. (D) The levels of cell cycle-related proteins were detected by Western blotting. The relative band intensities were quantitated after normalization to tubulin expression. The full size gels and blots were shown in the Supplementary Fig. S9 and band of interest is indicated with an arrow.