Cells were placed into the 96-well plates at the density of 4 × 104/mL in RPMI-1640. At the designated time point, the cells were coated with 100 μL sterile MTT (Sigma-Aldrich) in an incubator with 5% CO2 for 4 h at 37 °C. Afatinib was added with the desired drug treatment concentrations ranging from 0 to 20 μM and incubated for 72 h. The reaction waster was performed by removing the culture medium and then adding 100 μL of dimethyl sulfoxide (Sigma-Aldrich) for 0.5 h to dissolve the formaldehyde. Finally, absorbance values were measured at 490 nm. The IC50 (half-maximum inhibitory concentration) was used as the measure of relative cytotoxicity.

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