Cell Counting Kit-8 (CCK-8) assay

The C666-1 cells were seeded at a density of 3,000 cells/well in 96-well plates. After 24 h, the cell viability was determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc.), by measurement of the optical density at 450 nm (OD450) according to the kit protocol. Following this, a range of concentrations (10, 100, 500, 1,000 and 5,000 ng/ml) of S100P protein or FPS-ZM1 were added to the wells, an untreated control group was established, and the cells were cultured for 6 h at 37˚C. In subsequent experiments, the S100P protein and FPS-ZM1 were used at a concentration of 1,000 ng/ml, since at this concentration, the inhibition and proliferation rates were ~50%, respectively. Cell viability was assessed using the CCK-8 assay after incubation for 6, 12 and 24 h by adding 10 µl CCK-8 solution to each well, incubating for 30 min to 4 h at 37˚C according to the manufacturer's protocol, after which the OD450 value was measured.