MTT Assay and Determination of IC50

Cell proliferation was detected as described before (17). Briefly, cell line was seeded into a 96-well plate; after attachment, the cells were then treated with different doses of trametinib or bosutinib, alone or in combination for 24 h. After treatment, 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated with the cells for 3 h. After removing the medium, 100 μl of DMSO was added and then the absorbance at 570 nm was measured. The cell viability was normalized and is presented as a percentage of the control.

The interaction effect between bosutinib and trametinib was analyzed using the combination index (CI) (18, 19). The CI value is defined by the Loewe Additivity equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1 and (Dx)2 are the concentrations for D1 (bosutinib) and D2 (trametinib) alone that inhibit x% cell growth, and (D)1 and (D)2 are the concentrations of bosutinib and trametinib in combination that result in identical cell growth inhibition. Each CI value is the mean value of three independent experiments.