Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp

The Parkin‐siRNA lentivirus and control lentivirus were constructed by Genechem. 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinoline (DPQ) was from Apexbio (Houston, USA). Cyclosporin A and carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP) was from Selleck. N‐acetyl cysteine was from Beyotime (S0077). Anti‐PAR, Anti‐PARkin, anti‐COX IV, anti‐cyclophilin D (CypD) and anti‐translocator protein (TSPO) was from Abcam. Anti‐poly(ADP‐ribose) polymerase 1 (PARP‐1) was from Proteintech. Secondary antibodies for Western blotting were from Cell Signaling Technology.

NPCs were planted into culture plates at appropriate densities and cultured with a complete medium for 72 h. To evaluate the cytotoxicity of Se on NPCs, NPCs were treated with different concentrations of SS (0.5, 1, 3, 5, 7.5, 15, and 30 μM) (Sigma-Aldrich) or Se-Met (25, 50, 75, 100, 150, 300, and 500 μM) (MCE, NJ, USA) for 24 h. In addition, NPCs were treated with different concentrations of TBHP (25, 50, 75, 100, 125, and 150 μM) for 6 h to select the appropriate working concentration. To assess the effect of Se on the NPC viability under oxidative stress, NPCs were pretreated with different concentrations of SS or Se-Met for 24 h and then incubated with TBHP for another 6 h. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Selleck Chemicals, Houston, TX, USA) is an uncoupler of mitochondrial oxidative phosphorylation and an agonist of mitochondrial fission. To further explore the role of mitochondrial dynamics in TBHP-induced cell damages, NPCs were administrated with Se solely or in combination with 5 μM FCCP for 24 h and then treated with TBHP (100 μM) for another 6 h. Nrf2 inhibitor ML385 (10 μM) (Selleck Chemicals) was used to evaluate the role of the Nrf2 pathway in NPCs.