Fig. 5: PM exposure significantly elevates the mitochondrial respiratory and glycolytic rates in endometrial stem cells.

To evaluate the effect of PM exposure on the bioenergetic status of endometrial stem cells, the mitochondrial respiration and glycolytic rates were analyzed with or without PM exposure (a). Respiratory flux profiles of endometrial stem cells with or without exposure to PM (25 μg/ml) for 72 h were determined by collecting twelve consecutive oxygen consumption rate (OCR) measurements using a Seahorse XF analyzer. Cells were detached using trypsin-EDTA and reseeded in licensed cell culture miniplates at a density of 2 × 10⁴ cells per well in complete growth medium supplemented with 10% FBS. Cells were incubated overnight and washed 3 times with Seahorse XF medium. The ATP synthase inhibitor oligomycin (a complex V blocker, 1.5 μM) was added to inhibit ATP-coupled respiration. FCCP (a mitochondrial uncoupler, 2 μM) was added to decrease the mitochondrial membrane potential (Δψm). Rotenone (an inhibitor of complex I in the electron transport chain, 0.5 µM) and antimycin A (an inhibitor of complex III in the electron transport chain, 0.5 µM) were added to block mitochondrial respiration completely. The inhibitors were applied automatically in the analyzer, and the OCR was measured at 15-min intervals (b). PM exposure significantly increased the basal respiration rate (c), spare respiratory capacity (d), and maximal respiration rate (e) in endometrial stem cells. Overall ATP production in endometrial stem cells was significantly enhanced by PM exposure (f). The schematic of the procedure for real-time analysis of glycolysis in endometrial stem cells using the Seahorse XF analyzer is shown (g). For real-time analysis of glycolytic rates, the Seahorse XF glycolytic rate assay utilizes both ECAR (extracellular acidification rate) and OCR measurements to evaluate the glycolytic proton efflux rate (glycoPER) of the cells; in this assay, cells are incubated in glucose-free medium to which rotenone, antimycin A (1.67 μM), and finally 2-deoxyglucose (2-DG, glycolysis inhibitor, 50 mM) are sequentially added. The inhibitors were applied automatically in the analyzer, and the ECAR was measured at 15-minute intervals. The percentage of PER from glycolysis represents the contribution of the glycolytic pathway to the total ECAR (h). Compensatory glycolysis is the rate of glycolysis in cells following the inhibition of oxidative metabolism and refers to the ability of cells to drive compensatory energy production by using glycolysis to meet their energy demands (i). The ECAR values were normalized to the number of cells in each well. The bar graphs show the average of three independent experiments. Significant differences are presented. *p < 0.05, **p < 0.005, and ***p < 0.001 (two-sample t-test).