Fig. 4.

Inhibition of mTOR is critical for resveratrol’s suppression of Cd-induced apoptosis in neuronal cells. PC12 cells and primary neurons, or PC12 cells infected with lentiviral shRNAs to mTOR or GFP (as control), respectively, were pretreated with/without rapamycin (Rapa, 200 ng/ml) for 48 h and then resveratrol (Res, 100 μM) for 1 h, or pretreated with/without Res for 1 h, followed by exposure to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for TUNEL staining and live cell assay). A and D) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least five independent experiments. B and E) The relative densities for p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Thr70), cleaved-caspase-3 to β-tubulin were semi-quantified using NIH image J. C and F) Apoptotic cells were evaluated by in situ detection of fragmented DNA using TUNEL staining, and live cells were detected by counting viable cells using trypan blue exclusion. Results are presented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, difference vs control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001, difference vs 10 μM Cd group; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, ^$$$$p < 0.0001, difference vs Cd/Res group or Cd/Rapa group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p < 0.0001, mTOR shRNA group vs GFP shRNA group.