Cell viability Cell Counting kit (CCK)8 assay

HT29 cells in the exponential growth phase were seeded into 96-well plates at a density of 3×10³ cells/well, incubated for 24 h and then treated with increasing concentrations of AZD6244 (up to 10⁴ nM) with/without cetuximab (10 mg/l) for 72 h. Cell viability was assessed by adding 10 µl CCK-8 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) into the cell culture and incubating for an additional 2 h. The cell culture medium was then replaced with 200 µl DMSO, prior to measurements using a spectrophotometer at 450 nM. The cells were treated in triplicate and the experiments were repeated at least three times. The 50% inhibitory concentration (IC₅₀) values were then calculated using the median-effect equation derived by Chou (13).