Fig. 2. NAA promotes protein turnover affecting myotube diameter.

A Bright-field images of C2C12 myotubes. Scale bars, 50 µm. (n = 3). B The diameter measurement performed by ImageJ software. Data are expressed as mean ± SD of n = 3 independent experiments (*p < 0.05 vs 0 mM). C Western blot analysis of p-mTOR, mTOR and p-p70s6k (Thr389) levels in C2C12 myotubes. β-Actin was used as a loading control. D Western blot analysis of puromycin incorporation levels. Ponceau S was used as a loading control. Western blot analysis of E ubiquitinated protein levels and F BNIP3 and LC3 levels with/without NH₄Cl and leupeptin. β-Actin was used as a loading control. The Western blots reported are representative of three independent experiments that gave similar results. G The diameter measurement of bright-field images of myotubes MG132 or NH₄Cl/leupeptin treatment was performed by ImageJ software. Data are expressed as mean ± SD of n = 3 independent experiments (*p < 0.05, **p < 0.01 vs untreated).