Fig. 4.

Rapamycin blocks Cd-induced activation of JNK, Erk1/2 and p38 pathways by suppressing mitochondrial ROS induction in neuronal cells. PC12 and primary neurons were pretreated A and B) with/without rapamycin (0.2 μg/ml) for 48 h, or C and D) with/without rapamycin (0.2 μg/ml) for 48 h and then Mito-TEMPO (10 μM) for 1 h, followed by exposure to Cd (10 and/or 20 μM) for 24 h. A and C) Total cell lysates were subjected to Western blot analysis using indicated antibodies. The blots were probed for β-tubulin as a loading control. B and D) Similar results were observed in at least three independent experiments, and blots for p-JNK, p-c-Jun, p-Erk1/2, and p-p38 were semi-quantified. A and B) rapamycin potently suppressed Cd-induced phosphorylation of JNK/c-Jun, Erk1/2 and p38, and C and D) co-treatment with rapamycin/Mito-TEMPO exhibited a stronger inhibitory effect on Cd-induced activation of JNK/c-Jun, Erk1/2 and p38 than treatment with rapamycin or Mito-TEMPO alone in the cells. Results are presented as mean ± SEM (n = 3). ^a p < 0.05, difference with control group; ^b p < 0.05, difference with 10 μM Cd group; ^c p < 0.05, difference with 20 μM Cd group; ^d p < 0.05, difference with Cd/Mito-TEMPO group or Cd/Rapamycin group.