Clonogenic Assay

For clonogenicity assay, 1 × 10³/well adenovirus-infected A549 cells were seeded on 6-well plates. Cells were continuously cultured with refreshment of medium with or without nicotine with a 3-day interval for 10 days. For colony counting, the medium was removed and the cells were rinsed with PBS prior to being fixed with 4% paraformaldehyde at room temperature for 5 minutes. After removing the fixation solution, the cells were then stained with 0.5% crystal violet solution and incubated at room temperature for 30 minutes. The staining solution was carefully removed, and the cells were rinsed with H₂O to remove residual staining solution before they were air-dried at room temperature for up to 1 day. The number of colonies was counted and calculated under a light microscope. Each condition was tested in duplicate and each experiment was repeated for 3 times.