MTT assay

Cells were seeded at a density of 1.5 × 10⁴ cells in each well of 24-well plates and treated with erlotinib or KYA1797K for 0, 24, 48, and 72 hours. MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AMRESCO) was diluted in RPMI 1640 at a concentration of 0.5 mg/mL and was treated with cells for 2 hours at 37 °C. Media was removed and insoluble purple formazan was solubilized with 1 mL DMSO for 30 minutes. The absorbance of formazan product was determined at 590 nm. Each experiment was repeated three times. In order to acquire IC₅₀ values of KYA1797K treatment in different cell lines, the same procedure was performed except for the difference in cell density and treatment time. NCI-H1650, PC-9, NCI-H460, NCI-H23, and A549 cells were seeded at a density of 8.0 × 10³, 1.0 × 10⁴, 8.0 × 10³, 6.0 × 10³, and 1.5 × 10⁴ cells in each well of 96-well plates, respectively, and the all cells were treated with KYA1797K for 72 hours.