Fig. 3

Cigarette smoke exposure significantly increased metabolic activity (mitochondrial oxidative phosphorylation and glycolysis) in endometrial stem cells. To investigate the effect of cigarette smoke exposure on metabolic activity (energetic status) in endometrial stem cells, mitochondrial oxidative phosphorylation and glycolysis were measured after exposing or not exposing cells to cigarette smoke extract. Oxidative phosphorylation patterns of endometrial stem cells exposed or not to 1% cigarette smoke extract for 72 h were analyzed by measuring twelve consecutive oxygen consumption rates (OCR) using an XF analyzer (Seahorse Bioscience). A schematic of real-time analysis of mitochondrial oxidative phosphorylation using a Seahorse XF analyzer (A). Endometrial stem cells were seeded in multiplates at a density of 2 × 10.⁴ cells per well, incubated overnight in complete growth medium containing 10% FBS, and gently washed 3 times with seahorse XF medium. Cells were then treated with ATP synthase blocker oligomycin (a complex V inhibitor, 1.5 μM) to prevent coupled respiration (respiration linked to ATP production), FCCP (a potent uncoupler of mitochondrial oxidative phosphorylation, 2 μM) to reduce the electrochemical proton gradient (Δψm), rotenone (a complex I blocker of the electron transport chain, 0.5 uM), and antimycin A (a complex III blocker of the electron transport chain, 0.5 uM) to completely prevent mitochondrial oxidative phosphorylation. These blockers were added automatically into the multiplate, and OCR was analyzed every 15 min. Overall mitochondrial respiratory responses of endometrial stem cells were significantly elevated by cigarette smoke exposure and showed increased levels of nonmitochondrial oxygen consumption (B). Cigarette smoke exposure markedly enhanced the basal respiration rate (C), reserve respiratory capacity (D), and maximal respiratory potential (E). ATP synthesis was markedly increased by cigarette smoke exposure in mitochondria and cytosol (F). A schematic of cytosolic glycolysis analysis in real time using a Seahorse XF analyzer (G). The Seahorse XF glycolytic rate assay was used to measure OCR and ECAR (extracellular acidification rate) in real-time to determine glycolytic proton efflux rates (glycoPER) of endometrial stem cells, which were cultured in glucose-free medium after treatment with antimycin A (1.67 μM), rotenone, and 50 mM 2-deoxyglucose (2-DG, glycolysis blocker). These agents were added automatically to multiplates, and ECAR was analyzed at 15 min intervals. The overall percentage (%) of PER from glycolytic rate indicates the contribution made by glycolysis to total ECAR (G). Compensatory glycolysis is the glycolytic rate in cells after blocking oxidative phosphorylation and supporting compensatory energy synthesis through glycolysis to meet energy requirements. Cigarette smoke exposure also markedly increased basal glycolysis (H) and compensatory glycolysis (I). ECAR values were normalized with respect to cell counts in each well. Bar graphs represent the averages of three independent experiments. Significant differences are presented as *p < 0.05, **p < 0.005, and ***p < 0.001 (two-sample t test)