2.8. Cell culture

Hepa-1c1c7 cells obtained from American Type Culture Collection (Rockville, MD, USA) were cultured in Alpha minimum essential medium without nucleosides (AMEM; Life technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen), at 37 °C in a humidified atmosphere of 5% CO2. Hepatocytes were pretreated with BSO (500 μmol/L) for 16 h. BSO was then replaced with either normal medium or S-Nitroso-N-Acetyl-DL-Penicillamine (SNAP) (1 mmol/L) for 8 h in the absence or presence of MitoQ (0.5 μmol/L). After treatments, cells were washed and collected for further study. Cell variability was measured with Annexin Red Assay commercial Kit (Millipore, Billerica, MA), and the resultant fluorescence was detected with flow cytometer (Millipore, Billerica, MA).