Figure 2.

Long-term TS exposure-induced acquisition of the CSC-like phenotype. HBE cells were exposed to 2 % CSE (cigarette smoke extract) for 55 passages. Images of cell colonies (A) were taken and the number of colonies (B) was counted. (C) ZO-1, E-cadherin, Vimentin, and N-cadherin levels in TS-treated HBE cells were determined using A549 cells as positive controls. (D) Densitometric analyses of western blots of ZO-1, E-cadherin, Vimentin, and N-cadherin were performed following β-actin normalization. (E) 1 × 10⁶ non-CSE exposed control HBE cells, TS-exposed HBE cells, and A549 cells were subcutaneously injected in the front dorsum of nude mice and tumor incidence was analyzed 2 weeks later. (F) CHBE and TS-treated HBE cells were cultured in serum-free medium (SFM) for 7 days. Western blot analysis of lung CSC markers was then performed. (G) Densitometric analyses of western blots of CD133, ALDH1A1, Oct4, and Nanog were measured after β-actin normalization. Three independent experiments were performed. Data are expressed as the mean ± SD. Significance was assessed by one-way ANOVA or unpaired two-tailed Student's t tests. * P < 0.05, ** P < 0.01 compared to CHBE cells. THBE: HBE cells exposed to CSE for 55 passages; CHBE: HBE cells cultured under the same conditions for 55 passages, but not exposed to CSE. A549 cells were used as positive controls.