Fig. 5: Effects of VA on MAFbx and MURF1 expression in CM (CT26)-treated C2C12 cells.

A C2C12 myoblasts were incubated with VA at the indicated concentrations (0.1–100 μM) and cell viability after 24 h was assessed by a MTS assay. C2C12 myoblasts were differentiated in the absence or presence of CM (CT26). VA was treated at the indicated concentrations (10 μM and 100 μM). B mRNA expressions of Fbxo32, Trim63, and Myostatin were analyzed by Real-Time RT-PCR assays. Results were expressed relative to Gapdh. C Myofibers were immunostained with the antibody for MYH and counter-stained with DAPI for visualization of the cell nucleus (magnification ×100 and ×400). D Average diameter of C2C12 myofibers were measured using Fig. 5C. E Myofibers were stained with H&E (magnification ×100, scale bar = 275 μm). F, G Protein levels of F p4EBP1, MAFbx, MURF1, and G STAT3 were measured by western blot analysis. Results were expressed relative to GAPDH. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. CM (−); ^*p < 0.05 vs. CM (CT26). VA vanillic acid, CM (CT26) CT26-derived conditioned medium.