The stem cell-like properties of the side population fraction from A549 and non-small cell lung cancer cells were diminished following long-term passage. Clone formation was (A) photographed (magnification, ×1) and (B) calculated after 10 days culture. The formed spheres on day 14 were (C) photographed (magnification, ×4) and (D) counted to calculate the rate of formation. (E) Reverse transcription-quantitative polymerase chain reaction analysis of stemness genes expression (*P<0.05). NSCLC, non-small cell lung cancer; mRNA, messenger RNA; ABCG2, ATP-binding cassette sub-family G member 2; Bmi1, B lymphoma Mo-MLV insertion region 1 homolog; Oct4, octamer-binding transcription factor 4; Sox2, sex determining region Y-box 2.