Cell proliferation assay

The proliferation of 16HBE cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Inc.). 16HBE cells were seeded in 96-well plates (1.0×10⁴ cells/well), and were exposed to 0, 0.1 or 1 µM nicotine for 0, 48, 72 and 96 h at 37°C (31). Finally, 10 µl CCK-8 solution was added to 100 µl RPMI-1640 medium in each well for 2 h. Absorbance values were measured at a wavelength of 450 nm using an automated microplate reader (Molecular Devices, LLC).