Intracellular atp detection kit

Mm C was synthesized by Shanghai SIMR Biotech. Co., Ltd (Shanghai, China) with a purity of 95.65% (Supplementary Figure 6). MTT, sorafenib, reduced glutathione (GSH) detection kit, BCA kit for protein quantification, intracellular ATP detection kit and JC-1 MMP detection kit were from Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was from Nanjing KeyGEN Biotech. Co., Ltd. (Nanjing, Jiangsu, China). GSH, rotenone and cyclosporine A (Cs A) were from Solarbio Science & Technology Co., Ltd (Beijing, China). NAC was from Aladdin Biotech (Beijing, China). Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was from abcam (Cambridge, MA, USA). Valinomycin was from Sangon Biotech (Shanghai, China).

Cellular ATP content was measured following the manufacturers’ instructions (intracellular ATP detection kit, Beyotime). Briefly, Huh7.5 cells seeded in 12-well plate were incubated up to 3 or 6 h with GL22 or rotenone (positive control) at the concentrations indicated. Then, cells were rinsed three times with PBS and homogenized in ice-cold ATP lysis buffer. After centrifugation for 10 min at 4 °C, the supernatant of each sample was combined with equal volume of ATP assay buffer and then the level of chemiluminescence was immediately measured using a multi-detection microplate reader (Infinite M1000 Pro, TECAN). The ATP level of cells treated with DMSO in complete medium was considered as 100%.