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. 2022 Jun 23;15:896786. doi: 10.3389/fnmol.2022.896786

FIGURE 2.

FIGURE 2

Tumor necrosis factor-alpha increased mitochondrial respiration and reduced glycolysis in RPE. (A) Real-time measurement of oxygen consumption rate (OCR) using the Seahorse XFe96 BioAnalyzer to assess (B) OXPHOS parameters: basal respiration, ATP-linked respiration, proton leak, spare respiratory capacity (SRC), and maximal respiration based on responses to drug injections of oligomycin (Oligo), BAM15, and rotenone and antimycin A (Rot/AA) at 5 days with and without TNFα (n = 10–12, unpaired t-test) and (C) coupling efficiency. (D) Real-time measurement of extracellular acidification rate (ECAR) using the Seahorse XFe96 BioAnalyzer to assess glycolytic function parameters: (E) glycolysis, glycolytic capacity, and glycolytic reserve based on responses to drug injections of glucose (Glu), oligomycin (Oligo), and 2-deoxyglucose (2DG) at 5 days of treatment with and without TNFα (n = 10–12, unpaired t-test). (F) Intracellular ATP content at 5 days with or without TNFα (n = 6, unpaired t-test). (G) OCR curves and (H) ECAR curves for the Seahorse ATP rate assay at 5 days with TNFα. (I) Proportion of mitochondria-derived ATP production and glycolysis-derived ATP production and (J) increased ATP Rate Index representing a more oxidative and less glycolytic phenotype with TNFα treatment. (K) Real-time monitoring of OCR using the Resipher technology over 96 h following treatment with TNFα and (L) quantification of OCR over time (n = 7, unpaired t-test). Error bars are means ± SEM. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001; ns, not significant.