Fig. 3.

Rapamycin relieves Cd-induced mitochondrial ROS-dependent apoptosis in neuronal cells. PC12 and primary neurons were pretreated A and B) with/without rapamycin (0.2 μg/ml) for 48 h and then exposed to Cd (20 μM) in the presence or absence of TTFA (10 μM) or antimycin A (50 μM) for 24 h, or C-F) with/without rapamycin (0.2 μg/ml) for 48 h, Mito-TEMPO (10 μM) for 1 h and then exposed to Cd (20 μM) for 24 h, followed by A-C) ROS imaging using an oxidant-sensitive probe CM-H₂DCFDA, D) cell apoptosis analysis using DAPI staining, or E) Western blotting using the indicated antibodies. The blots were probed for β-tubulin as a loading control. F) Similar results were observed in at least three independent experiments, and blots for cleaved-caspase-3, and cleaved-PARP were semi-quantified. A) Co-treatment with rapamycin/TTFA inhibited Cd-evoked ROS fluorescence more potently than treatment with rapamycin or TTFA alone in the cells. B) Treatment with antimycin A alone markedly elevated ROS and strengthened Cd-increased ROS levels, which were repressed by rapamycin pretreatment. C-F) Pretreatment with Mito-TEMPO obviously inhibited Cd-induced ROS levels and cell apoptosis, and dramatically potentiated the inhibitory effects of rapamycin on Cd-induced the events in PC12 cells and primary neurons. Results are presented as mean ± SEM (n = 3–5). ^a p<0.05, difference with control group; ^b p<0.05, difference with 20 μM Cd group; ^c p<0.05, difference with Cd/TTFA group, Cd/Antimycin A group, Cd/Mito-TEMPO group or Cd/Rapamycin group.