Cell Counting Kit-8 (CCK8) Assay

ACHN and 786-O cells had been transfected with plasmids or siRNAs for 48 h before subsequent experimentation. Cells were inoculated on 96-well plates at a cell density of 1×10³ cells per well with 100 µl of medium. A cell proliferation assay was performed using Cell Counting Kit-8 (CCK8; MedChemExpress, USA) at a concentration of 10μl in 100μl serum-free medium every 24 h for four days according to the manufacturer’s protocols. After incubation for 2 h at 37°C, the optical density of each well was measured at 450 nm with a spectrophotometer to measure the quantity of living cells. Finally, the absorbance of cells over four days were plotted in a graph for a reflection of cell proliferation rate.