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. 2019 Apr 4;30(15):1775–1796. doi: 10.1089/ars.2018.7514

FIG. 9.

FIG. 9.

STC1 maintains mitochondrial bioenergetics. (A–C) Comparison of mitochondrial function of WT and TLR4−/−. (A) OCR was immediately measured using the Seahorse XF96 Analyzer. Kinetic OCR responses to oligomycin (1 μM), FCCP (5 μM), rotenone (1 μM), and antimycin (1 μM) were recorded. (B) Fundamental parameters of mitochondrial function including basal respiration, proton leak, ATP-production-related oxygen consumption, maximum respiration, and nonmitochondrial oxygen consumption rates were normalized based on number of live cells. (C) Mitochondrial coupling efficiency was calculated as the percentage of ATP production to basal mito-OCR (as ATP production/basal mito OCR × 100%). *p < 0.05 vs WT, n = 10–20. (D–F) WT MLECs were treated with lentivirus (lenti-Ctrl or lenti-STC1sh) and TLR4−/− MLECs were treated with lentivirus (lenti-Ctrl or STC1 overexpression lenti-STC1oe). Mitochondrial bioenergetic profile of MLECs was studied using the “Mito stress kit,” Seahorse Biosciences. (D) A representative graph output from XF96 showing the OCR response to oligomycin, FCCP, rotenone, and antimycin in normoxia. (E) Fundamental parameters of mitochondrial function including basal respiration, proton leak, ATP-production-related oxygen consumption, maximum respiration, and nonmitochondrial oxygen consumption rates were normalized based on number of live cells. (F) Mitochondrial coupling efficiency. A minimum number of n = 3 with 10–20 replicate wells per group was employed for all experiments. *p < 0.05 vs WT lenti-Ctrl; **p < 0.05 vs TLR4−/− lenti-Ctrl. Data were all normalized to 10,000 cells, using cell nuclei staining with Hoechst 33342 upon completion of Seahorse measurements. Real-time OCR were recorded and normalized with cell count using Wave Desktop 2.6. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for pairwise comparisons in (A) and post hoc Tukey's HSD test calculator for multiple comparisons in (B–F). ATP, adenosine triphosphate; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; OCR, oxygen consumption rate.