Fig. 9.

mTORC2-mediated Akt pathway contributes to resveratrol’s inhibition of Cd-induced Erk1/2 activation, ROS generation and apoptosis in neuronal cells. PC12 cells and primary neurons, or PC12 cells infected with Ad-dn-Akt, Ad-myr-Akt or Ad-LacZ (as control), respectively, were pretreated with/without Akt inhibitor X (20 μM) for 1 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 ROS imaging, live cell assay and TUNEL staining). A and F) 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 and G) The relative densities for p-Akt (Ser473), p-Akt (Thr308), p-GSK3β (Ser9), p-S6K1 (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Thr70), p-Erk1/2 (Thr202/Tyr204), cleaved-caspase-3 to β-tubulin were semi-quantified using NIH image J. C, D and H) Cell ROS was imaged and quantified using an oxidant-sensitive probe CM-H₂DCFDA. Scale bar: 20 μm. E and I) 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/Akt inhibitor X group or Cd/Res group. ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p < 0.0001, Ad-dn-Akt group or Ad-myr-Akt vs Ad-LacZ group.