Fig. 3.

Pharmacological inhibition of NOX2 with apocynin or DPI, or knockdown of NOX2 prevents activation of AMPK, inhibition of Akt/mTOR, generation of H₂O₂, and apoptosis in PD toxins-induced neuronal cells. PC12 cells and primary neurons, or PC12 cells infected with lentiviral shRNA to NOX2 or GFP (as control), respectively, were treated with/without 6-OHDA (120 μM), MPP⁺ (1 mM) or rotenone (1 μM) for 24 h following pre-incubation with/without apocynin (50 μM) or DPI (10 μM) for 1 h, or treated with/without 6-OHDA (120 μM), MPP⁺ (1 mM) or rotenone (1 μM) for 24 h. 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 three independent experiments. B, E and H Intracellular H₂O₂ was imaged and quantified using a peroxide-selective probe H₂DCFDA. C, F and I Apoptotic cells were evaluated by nuclear fragmentation and condensation using DAPI staining. Results were presented as mean ± SEM, n = 5. ^aP < 0.05, difference with control group; ^bP < 0.05, + Apocynin group vs - Apocynin group; ^cP < 0.05, + DPI group vs - DPI group; ^dP < 0.05, NOX2 shRNA group vs GFP shRNA group.