Fig. 5.

Inhibition of mTORC1/2, Erk1/2 or JNK by PP242, U0126 and/or SP600125 strengthens resveratrol’s protection against Cd-induced apoptosis in neuronal cells. PC12 cells and primary neurons were pretreated with/without PP242 (1 μM), U0126 (5 μM) and/or SP600125 (20 μM) for 1 h and then resveratrol (Res, 100 μM) for 1 h, followed by exposure to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for TUNEL staining). A, D and G) 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, E and H) The relative densities for p-Akt (Ser473), p-Akt (Thr308), p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Thr70), p-Erk1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-c-Jun (Ser63), cleaved-caspase-3 to β-tubulin were semi-quantified using NIH image J. C, F and I) Apoptotic cells were evaluated by in situ detection of fragmented DNA using TUNEL staining. 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, Cd/PP242 group, Cd/U0126 group or Cd/SP600125 group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p < 0.0001, difference vs Cd/Res/PP242 group, Cd/Res/U0126 group or Cd/Res/SP600125 group.