Fig. 1

Cigarette smoke exposure significantly reduced the proliferations and migrations of endometrial stem cells and induced cellular senescence. We postulated that cigarette smoke exposure suppresses various tissue regeneration-associated functions of endometrial stem cells, such as proliferation, senescence, migration potential, and differentiation capacity, and pluripotency/stemness (A). The inhibitory effects of cigarette smoke extract (0.5, 1, 3, 5%) on the self-renewal capacity of endometrial stem cells were analyzed using a 72-h MTT assay. Proliferative potentials (%) were calculated by expressing numbers of viable cells after treatment with cigarette smoke extract (0.5, 1, 3, 5%) as percentages of numbers after treatment with vehicle (B). Effects of cigarette smoke exposure on the senescence (aging) of endometrial stem cells were assessed by analyzing senescence-associated β-galactosidase (SA-β-Gal) activity in cells consecutively exposed to 1% cigarette smoke extract treatment for 72 h (C). Effects of cigarette smoke exposure on the mRNA levels of various senescence (aging) markers (p16, p18, p21, and IL-6) were determined by real-time PCR (D). The GEO (Gene Expression Omnibus) metadata respiratory (https://www.ncbi. nlm.nih.gov/geo/) was analyzed to assess relationships between cigarette smoking and levels of various senescence (aging) markers (E). Endometrial stem cells were exposed to 1% cigarette smoke extract for 72 h, and inhibitory effects on migration were determined using transwell assays (F). The relative protein levels of key regulators of cell migration (MMP-2/9) after exposure or not to cigarette smoke exposure were analyzed by western blotting (G). β-actin was used as the internal control for western blotting, and PPIA was used as the housekeeping gene real-time PCR. All experiments were performed in triplicate. Data are presented as means ± standard deviations (SDs). *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)