Cell counting kit 8 (cck8)

Cell viability was determined using a cell counting kit-8 (CCK-8, Sparkjade, Shandong, China). HepG2 cells were seeded into 96-well plates at a density of 5×10³, adhered overnight, and treated with different concentrations of SYTZD drug containing serum for 24 h. Afterwards, 10 µl of CCK-8 solution was added to each well of the 96-well plate, and the cells were incubated at 37°C for 2–3 h. Finally, the absorbance value of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).

UP302 was purchased from Nanjing m&m biotechnology Co. Ltd. The following reagents were used: fetal bovine serum (Procell, China), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium(IMDM), trypsin (Servicebio, China), CCK8 (Sparkjade, China), chloroquine (Sparkjade, China), PI and FITC, Mitochondrial membrane potential assay kit with JC-1 and Reactive Oxygen Species Assay Kit (Beyotime, China), Antibodies against Caspase-3, BAX, BCL-2, LC3B, AKT123, PI3K, P-PI3K, P-AKT, AIF, Tomm20, PINK1, Parkin, Beclin1, and β-actin were purchased from ABclonal(China). Twelve Balb/c female nude mice, aged four weeks, were purchased from Jinan Pengyue Experimental Animal Breeding Co. LTD.

CCK8 (CT0001-B, Sparkjade, Jinan, China) was used to test the inhibitory effect of celastrol on B16-F10 cell proliferation. B16-F10 cells were seeded into 96-well plates at a density of 6 × 10³ cells/well. After overnight incubation, different concentrations (0, 0.03, 0.3, 1, 3, and 10 μM) of celastrol were added to cells for 12, 24, 36, and 48 h, and then cells of each well were treated with CCK-8 solution for 1 h at the end of the incubation period. A wavelength of 450 nm was selected, and an enzyme-linked immunosorbent assay was used to detect the light absorption value of each well.