Fig. 3.

Cd-induced activation of Akt/mTOR pathway and cell apoptosis are attenuated by simultaneous or late treatment with resveratrol in neuronal cells. PC12 cells and primary neurons were co-treated with/without resveratrol (100 μM)/Cd (10 and 20 μM) simultaneously for 4 h (for Western blotting) or 24 h (for TUNEL staining and annexin-V-FITC/PI staining), or pretreated with/without Cd (10 and 20 μM) for 1 h followed by exposure to resveratrol (100 μM) for 24 h. A) Resveratrol powerfully blocked Cd-evoked p-Akt, p-mTOR, p-S6K1, p-S6, p-4E-BP1 and cleaved-caspase-3 in PC12 cells and primary neurons. Total cell lysates were subjected to Western blot analysis 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) The relative densities for p-Akt (Ser473), p-Akt (Thr308), 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 E) IOD values of TUNEL-positive cells were evaluated by in situ detection of fragmented DNA using TUNEL staining. D and F) Quantitative analysis of apoptotic cells was determined by FACS using annexin-V-FITC/PI 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 20 μM Cd group.