Shifting anaerobic to aerobic metabolism stimulates apoptosis through modulation of redox balance: potential intervention in the pathogenesis of postoperative adhesions  Nicole M. Fletcher, Ph.D., Awoniyi O. Awonuga, M.D., Bailey R. Neubauer, B.S., Mohammed S. Abusamaan, M.D., Mohammed G. Saed, B.A., Michael P. Diamond, M.D., Ghassan M. Saed, Ph.D.  Fertility and Sterility  Volume 104, Issue 4, Pages 1022-1029 (October 2015) DOI: 10.1016/j.fertnstert.2015.06.041 Copyright © 2015 Terms and Conditions

Figure 1 Real-time RT-PCR for iNOS mRNA levels and NO levels in normal peritoneal and adhesion fibroblasts. Normal peritoneal and adhesion fibroblasts were treated with increasing doses of DCA (0, 20, 40, and 80 μg/mL) for 24 hours. (A) Real-time RT-PCR was used to measure iNOS mRNA levels. (B) Nitric oxide levels were detected using the Greiss assay, which measures nitrate/nitrite levels, in culture media. All experiments were performed in triplicate. *P<.05 vs. normal fibroblast controls; **P<.05 vs. adhesion fibroblast controls. Fertility and Sterility 2015 104, 1022-1029DOI: (10.1016/j.fertnstert.2015.06.041) Copyright © 2015 Terms and Conditions

Figure 2 Real-time RT-PCR for SOD3 in normal peritoneal and adhesion fibroblasts. Real-time RT-PCR was used to measure SOD3 mRNA levels in normal peritoneal and adhesion fibroblasts, treated with increasing doses of DCA (0, 20, 40, and 80 μg/mL) for 24 hours. All experiments were performed in triplicate. *P<.05 vs. controls; ** P<.05 vs. adhesion fibroblast controls. Fertility and Sterility 2015 104, 1022-1029DOI: (10.1016/j.fertnstert.2015.06.041) Copyright © 2015 Terms and Conditions

Figure 3 Detection of apoptosis in normal peritoneal and adhesion fibroblasts. (A) Caspase-3 activity was measured in cell lysates from normal peritoneal and adhesion fibroblasts, treated with increasing doses of DCA (0, 20, 40, and 80 μg/mL) for 24 hours. All experiments were performed in triplicate. *P<.05 vs. controls; ** P<.05 vs. adhesion fibroblast controls. (B) The amount of DNA fragmentation (apoptosis) was assessed by the TUNEL assay after DCA treatment (80 μg/mL, 24 hours) as compared with control cells. Apoptotic cells were visualized (63×) with fluorescein-12-dUTP (green). Experiments were performed in triplicate. Fertility and Sterility 2015 104, 1022-1029DOI: (10.1016/j.fertnstert.2015.06.041) Copyright © 2015 Terms and Conditions

Figure 4 Detection of mitochondrial membrane potential (Δψm) in normal peritoneal and adhesion fibroblasts. Fibroblasts were treated with hypoxia with or without DCA for 24 hours using the JC-1 Mitochondrial Membrane Potential (Δψm) Kit. High mitochondrial Δψm is represented by intense red fluorescence, and low Δψm is represented by green fluorescence. The fluorescence was detected with an Axiovert 25 inverted microscope (Zeiss) using fluorescein isothiocyanate conjugate (green), and Texas Red (red) fluorescent filters with excitation and emission wavelengths of 470 and 525, 596 and 613 nm, respectively. Fertility and Sterility 2015 104, 1022-1029DOI: (10.1016/j.fertnstert.2015.06.041) Copyright © 2015 Terms and Conditions