Fig. 7: Abolished effect of VA on the improvement of muscle loss in siSirt3 and CM (CT26)-treated C2C12 cells.

A After treatment with siSirt3 (10 pM) in C2C12 cells, the SIRT3 protein level was measured by western blot analysis. Results were expressed relative to GAPDH. B Myofibers were immunostained with the antibody for SIRT3 and counter-stained with DAPI for visualization of the cell nucleus (magnification ×400). C Myofibers were stained with H&E (magnification ×100, scale bar = 275 μm). Average diameter of C2C12 myofibers were measured. D The mRNA levels of Sirt3, Ppargc1a, Nfe2l2, Mfn1, and Opa1 were analyzed by Real-Time RT-PCR. Results were expressed relative to Gapdh. E Myofibers were immunostained with MitoTracker and counter-stained with DAPI for visualization of cell nucleus (magnification ×400). F Fluorescence intensity for MitoTracker was quantified using ImageJ software and presented as a bar graph. G Myofibers were immunostained with antibodies for MURF1 and counter-stained with DAPI for visualization of cell nucleus (magnification ×400). H Fluorescence intensity for MURF1 was quantified using ImageJ software and presented as a bar graph. 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 (CT26); ^&p < 0.05 vs. CM (CT26) with VA. VA vanillic acid, CM (CT26) CT26-derived conditioned medium.