MTT assay

NSCLC proliferation was examined using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay. Cells (180 μl) in the exponential growth phase (3 × 10³ cells/well) were seeded into a 96-well plates, and 20 μl of gefitinib and/or PD 0332991 were added. After incubation at 37°C, 20 μl of MTT solution (5 mg/ml in PBS) was added to each well, and the plates were incubated for an additional 4 hr at 37°C. The plates were centrifuged at 200 x g for 5 min, medium was aspirated from each well, and 180 μl of DMSO was added to each well to dissolve the formazan. Optical density was measured at 490 nm with a SoftMax Pro analysis program interfaced with a SpectraMax M5 Microplate reader (Molecular Devices, Sunnyvale, CA, USA). Each experiment was carried out in 6 replicate wells for each concentration and in 3-4 independent experiments. The IC50-value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on survival curves.