Fig. 2

Cigarette smoke exposure significantly reduced differentiation capacity and the pluripotency/stemness of endometrial stem cells. Human endometrial stem cells were pretreated with or without 1% cigarette smoke extract and then cultured in adipocyte or osteoblast differentiation medium. The suppressive effects of cigarette smoke exposure on adipocyte (A) and osteoblast (B) differentiation were analyzed by oil red O and alizarin red staining, respectively. The relative quantification of calcium mineral contents and lipid droplet formation within differentiated cells was determined by measuring absorbance at 500 nm and 570 nm, respectively. The suppressive effects of cigarette smoke exposure on the mRNA levels of various pluripotency-associated genes (C-MYC, KLF4, NANOG, OCT4, and SOX2) were analyzed by real-time PCR (C). The GEO metadata respiratory was analyzed to investigate the relationships between cigarette smoke exposure and levels of various pluripotency-associated genes (D). PPIA was used as the housekeeping gene for real-time PCR. All experiments were performed in triplicate, and data are presented as means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test)