## Results
### Effect of RE on AICAR-Induced C2C12 Myotube Atrophy  
Before evaluating whether RE promoted hypertrophy in AICAR-induced C2C12 myotube atrophy, the cytotoxicity of simultaneous RE and AICAR treatment was evaluated using maximum RE and AICAR concentrations (40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 1 mM, respectively). After no serious cytotoxicity was observed, RE concentrations of up to 40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL were used (Figure 4). C2C12 myoblasts were cultured in differentiation medium for 6 d, and the resulting myotubes were treated with 1 mM AICAR for 24 h to induce muscle cell atrophy. Then, various concentrations of RE were added, and the inhibition of muscle cell atrophy and promotion of hypertrophy were evaluated after 24 h.  
The C2C12 myoblasts clearly differentiated into myotubes when cultured in differentiation medium, with or without 40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL RE (Figure 5a), and clearly exhibited muscle cell atrophy when treated with 1 mM AICAR. However, treatment with RE reduced AICAR-induced muscle cell atrophy in a concentration-dependent manner and resulted in muscle cell hypertrophy (Figure 5a). Treatment with AICAR alone reduced myotube diameter by \(\sim\)60% when compared with vehicle-treated control myotubes (Figure 5b). In addition, myotube diameter increased with increasing RE concentration, reaching 70-80% the diameter of vehicle-treated control myotubes at 40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL RE (Figure 5b). Figure 3: Effects of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on C2C12 myotube viability and differentiation. C2C12 cells were treated with indicated concentrations of AICAR and cultured for 24 h; cell counting (**a**) and MTT assay (**b**) were performed. Values and error bars indicate means \(\pm\) SD of three separate experiments. Asterisks indicate significant differences between RE- and vehicle-treated cells before differentiation (ANOVA; *, \(p<0.05\); **, \(p<0.01\); ***, \(p<0.001\)); (**c**) Phase-contrast micrographs of C2C12 cells before and after 6 d of differentiation when cultured with different concentrations of AICAR. GM: growth medium. \(200\times\) magnification; (**d**) C2C12 cell diameter before and after 4 d of differentiation when cultured with different concentrations of AICAR. Values and error bars indicate means \(\pm\) SD of three separate experiments. ***, significant difference between RE- and vehicle-treated cells before differentiation (ANOVA, \(p<0.001\)); ***, significant difference between RE- and vehicle-treated cells after differentiation (ANOVA, \(p<0.01\)); ***, significant difference between RE- and vehicle-treated cells after differentiation (ANOVA, \(p<0.001\)). \(p<0.001\). Figure 4: Effects of rosemary extract (RE) on the viability of 5-aminoimidazole-4-carboxamide ribonuclease(AICAR)-treated C2C12 myoblasts: C2C12 myoblasts were treated with AICAR (1 mM) and indicated concentrations of RE, cultured for 24 h, and then evaluated using the MTT assay. Values and error bars indicate means \(\pm\) SD of three separate experiments. *, significant difference between RE- and vehicle-treated cells before differentiation (ANOVA, \(p\) < 0.05); **, significant difference between RE- and vehicle-treated cells before differentiation (ANOVA, \(p\) < 0.01); ##, significant difference between RE- and AICAR-treated cells before differentiation (ANOVA, \(p\) < 0.01). Figure 5: Effects of rosemary extract (RE) on 5-aminoimidazole-4-carboxamide ribonuclease(AICAR)-induced muscle cell atrophy in C2C12 myotubes. C2C12 cells were cultured in a differentiation medium for 6 d, until fully differentiated, cultured with or without 1 mM AICAR for 24 h, and then cultured with or without RE for an additional 24 h. (**a**) Phase-contrast micrographs of C2C12 cells after differentiation when cultured with AICAR and/or RE. 200\(\times\) magnification; (**b**) C2C12 myotube diameter when differentiated with AICAR and/or RE. Values and error bars indicate means \(\pm\) SD of three separate experiments.