Oligomycin

Norbergenin (purity ≥ 98%) was obtained from Baoji Herbest Company (Baoji Herbest Bio-Tech Co., Ltd, Shanxi, China), LPS from Escherichia coli 055:B5 (Cat: L2880), Antimycin A (Cat: A8674), sodium pyruvate (Cat: 11360039), D-(+)-Glucose (Cat: G7021), D-glutamine (Cat: 158968) and 2-Deoxy-D-glucose (2-DG, Cat: D8375) were purchased from Sigma-Aldrich. SB203580 (Cat: HY-10256), U0126 (Cat: HY-12031), SP600125 (Cat: HY-12041), oligomycin (Cat: HY-N6782), rotenone (Cat: HY-B1756), FCCP (Cat: HY-50202), methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Cat: HY15924) were obtained from MedChemExpress. Griess Reagent System (Cat: G2930) was obtained from Promega Corporation. Transfer membrane Immubilon-P PVDF (Cat: IPVH00010) was obtained from Millipore. All other reagents were of analytical grade and obtained from Sigma.

661w cells were purchased and validated from Aolu Biotechnology (Shanghai, China). The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. After the cell confluence reached 80%, the cells were treated with 0–10 mmol/L hydrogen peroxide (H₂O₂; Amresco, OH, USA, Cat#E882). For the group treated with 1 mmol/L nicotinamide riboside (NR; Yuanye, Shanghai, China, Cat#S31692) or 10 μmol/L doxycycline (DOX; MedChem Express, NJ, USA, Cat#HY-N0565B) and H₂O₂, the cells were pretreated with NR or DOX for 24 h, then incubated with H₂O₂ for additional 12 h. For the group treated with 50 μmol/L chloroquine (CQ; MedChem Express, Cat#HY-17589) or 1 μmol/L cyclosporine (CsA; MedChem Express, Cat#HY-B0579), the cells were pretreated with CQ or CsA for 2 h before NR treatment. Incubation of 10 μg/mL oligomycin (MedChem Express, Cat#HY-N6782) and 1 μmol/L antimycin A (GLPBIO, CA, USA, Cat#GC42818) for 6 h was used as a positive control of mitophagy. NR, CsA, oligomycin, and antimycin A were dissolved in dimethysulfoxide (DMSO), while DOX and CQ were dissolved in distilled water. All the drugs were further diluted in the cell culture medium to achieve their working concentrations.