MTT assay

A total of 3×10³ NCI-H1299 and LLC cells per well were seeded onto 96-well plates (cat no. 3599; Corning Incorporated, Corning, NY, USA) and treated with 100 µl of CMF at 0.3, 1, 3, 10, 30, 90 µ1/ml in NCI-H1299 cells and 0.03, 0.1, 0.3, 0.9 µg/ml in LLC cells. In a pre-screening experiment (data not shown), the effect of CMF was stronger against LLC cells than against NCI-H1299 cells. Therefore, the two cell lines were treated with different concentrations of CMF to obtain the IC₅₀ values. The same volume (100 µl) of corresponding complete medium was used as a negative control. Following incubation for 24, 48, 72 h at 37°C, 5% CO₂, 20 µl MTT solution (5 mg/ml) was added into each well. Then 200 µl DMSO was used to dissolve the formazan crystals and optical density (OD) absorbance was measured at 570 nm using a 96-well microplate reader. The results were presented as cell viability (%)=OD_treatment/OD_ncx100% and 50% inhibitory concentration (IC₅₀) was calculated by linear-regression analysis. All experiments were performed in triplicate.