Cell viability and drug combination assay

Cells were cultured in 96-well plates (2000 cells/well) for 24 h, and were treated with the indicated concentrations (range from 10 to 10⁵ nM)of CB839, selumetinib, or both drugs in combination for 72 h. Cell viability was measured using the Cell Counting Kit 8 (CCK8, Dojindo Laboratories, Tokyo, Japan) and microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at the 450 nm wavelength. The half maximal inhibitory concentration (IC₅₀) and 10% inhibitory concentration (IC₁₀) were calculated by Probit model. Combination index (CI) data were obtained using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). CI values <1 indicated a synergistic drug-drug interaction, while CI values <0.3 indicated a strong synergistic effect.