Autophagy inhibition by autophagy-related protein 5 (atg5) knockdown aggravated bupivacaine myotoxicity in C2c12 cells. (A) atg5 messenger RNA (mRNA) levels after small interfering RNA (siRNA). C2c12 cells were transfected with atg5 siRNA. Cells transfected with scrambled RNA served as negative control (NC). atg5 mRNA levels were analyzed by real-time quantitative polymerase chain reaction 24 h after siRNA transfection. * P < 0.01 versus NC group, n = 4 per group. (B) atg5 protein levels, microtubule-associated protein light chain 3 (LC3) conversion and p62 protein levels after siRNA. C2c12 cells were collected 48 h after siRNA transfection. Cellular extracts were then prepared for Western blot with the indicated antibodies. The same membrane was then probed with an antibody against α-tubulin as a loading control. *P < 0.01 versus the respective NC group, n = 4 per group. (C) Cell survival. C2c12 cells were treated with bupivacaine (600 μM) at 48 h after atg5 siRNA. Cell viability was examined in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay 24 h after bupivacaine exposure. * P < 0.01. n = 4 per group.