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. 2017 Nov 7;7:23–34. doi: 10.1016/j.molmet.2017.11.002

Figure 4.

Figure 4

OxPLs redirect TCA metabolism and inhibit macrophage respiratory capacity. A. Intracellular α-ketoglutarate from metabolomics of M0 (vehicle, RPMI media), Mox (10 μg/mL OxPAPC), M1 (1 μg/mL LTA), and M2 (10 ng/mL IL4) BMDMs (6 h) represented by box and whisker plots (n = 5). B. Intracellular succinate from metabolomics of M0, Mox, M1, and M2 BMDMs (6 h) represented by box and whisker plots (n = 5). C. mRNA expression of nuclear-encoded complex II subunit, Sdhaf2, as measured by qPCR in BMDMs treated with vehicle or 10 μg/mL OxPAPC for 4 h (n = 4). D. Fold change of intracellular succinate dehydrogenase inhibitor, methylmalonate, in Mox BMDMs compared to M0 as found by metabolomics (n = 5). E. The tri-carboxylic acid (TCA) cycle in Mox, M1, and M2 BMDMs, highlighting metabolites significantly changed from M0 (n = 5). F. Mitochondrial stress test (MST) of BMDMs treated with vehicle (RPMI media) or 10 μg/mL OxPAPC for 4 h (n = 4). The oxygen consumption rate (OCR) was measured initially (basal; red), and after injection of 1 μM oligomycin, 2 μM of the uncoupler BAM15 (maximal; teal), and 10 μM antimycin A & 1 μM rotenone (AA/Rot; gray; non-mitochondrial). Basal and maximal OCR were calculated by subtracting the mean OCR of the first three (basal) or post-BAM15 (maximal) measurements from the mean OCR of the post-AA/Rot measurements. G. MST of BMDMs treated with vehicle (RPMI media) or 30 μg/mL OxPAPE for 4 h (n = 4). H. Basal and maximal oxygen consumption rate (OCR) measured in BMDMs treated acutely with vehicle, 1 μg/mL LTA, or 10–100 μg/mL OxPAPC. Treatment was injected into wells containing BMDMs after 30 min of unstimulated measurement. OCR was measured for 2 h (basal) after which 2 μM BAM15 was injected to assess maximal OCR. Antimycin A and rotenone were injected to determine the non-mitochondrial OCR (background). Data represented as %OCR, where 100% refers to the unstimulated basal OCR (n = 4). I. MST of BMDMs treated with vehicle (RPMI media) or 10 ng/mL IL4 for 16 h, followed by vehicle (RPMI media) or 10 μg/mL OxPAPC for 4 h (n = 4). Data are expressed as mean ± SEM. Biological replicates indicated by (n). Statistical significance calculated by Welch's 2-sided t-test (*p ≤ 0.05; **p < 0.01; ***p < 0.001).