Dulbecco s modified eagle s medium

Fetal bovine serum (FBS), trypsin, and Hank's solution were purchased from Gibco (Grand Island, NY, USA). Dulbecco's Modified Eagle's Medium (DMEM), Hoechst 33342, tetramethylrhodamine ethyl ester (TMRE), calcein-AM, and Cyclosporin A (CSA) were brought from KeyGEN Biotech (Nanjing, China). Mito-Tracker Red, Mito-Tracker Green, and Mito-Sox Red were obtained from Invitrogen (Carlsbad, CA, USA). Chir99021 was purchased from Selleck Chemicals (Houston, TX, USA). Edaravone, Mdivi-1, and Mito-TEMPO were from Sigma (St Louis, MO, USA). These reagents were dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration was <0.1% v/v. Primary antibodies and secondary antibodies for western blot analyses are listed in Supplementary Tables 1 and 2. Cytochrome c ELISA kit was purchased from Cusabio (Wuhan, China) and the intracellular calcium kit was from Beyotime Biotechnology (Nanjing, China).

Human CRC cell lines SW480, HCT116, SW620, LOVO, DLD1, and NCM460 were purchased form the American Type Culture Collection (Manassas, Virginia, USA). The cells were cultured at 37 °C in a 5% CO₂ incubator in Dulbecco’s modified Eagle’s medium (KeyGEN BioTECH, Jiangsu, China) with 10% fetal bovine serum (Gibco, USA). Lentivirus-HPSE2 (LV-HPSE2) were constructed in the lentivirus vector GV492 by a commercial service (Genechem Biotech Inc, Shanghai, China), while lentivirus vector GV492 was adopted as the control. LV-HPSE2 was packaged in 293 T cells. The supernatant of cell culture medium containing lentivirus granules was collected and the viral titer of the virus solution was determined. HCT116 and SW480 with a growth fusion degree of about 80% were digested by trypsinase and the cell suspension was prepared. Cells were seeded into six-well plates at 1.0 × 10⁶ cells per well and maintained at 37℃ in a humidified atmosphere of 5% CO₂. Cells were divided into two groups, one group was added with 10 ml virus solution and the other group was added with 10 ml empty vector virus solution. The multiplicity of infection (MOI_HCT116 and MOI_SW480) was 10. After 16 h of cell culture, the culture medium was replaced. After 3 days of infection, the cells were in good condition. The positive clones were screened by using a complete culture medium containing 2.0 mg/mL puromycin for 4 weeks.

The human HCC cell line (PLC/PRF/5) and mouse HCC cell line (Hepa1-6) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (KeyGEN, Nanjing, China) containing 10% fetal bovine serum (FBS) (Sigma, Saint Louis, USA) and 100 U/mL of penicillin and 50 μg/mL streptomycin (Gibco, California, USA) in a 37 °C humidified incubator with 5% CO₂.

The Chinese National Collection of Authenticated Cell Cultures (Shanghai, China) provided two human lung adenocarcinoma cell lines, A549 and HCC827, as well as human embryonic kidney cell line 293T, which were cultured in high-glucose Dulbecco’s modified Eagle’s medium (KeyGEN, Nanjing, Jiangsu, China) with 10% fetal bovine serum (Every Green, Huzhou, Zhejiang, China), 100 U/mL penicillin (Beyotime, Shanghai, China), and 0.1mg/ml streptomycin (Beyotime).