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. 2022 Feb 4;50:102257. doi: 10.1016/j.redox.2022.102257

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 6 — Extracellular vesicles from PKM2-activated T lymphocytes contain more PUFA-containing PLs, which may provide substrates for lipid peroxidation in target macrophages.

HPLC-MS/MS analysis of lipid metabolites in different T lymphocyte-derived EVs isolated from culture supernatants. (A) PCA scatter plot of the lipid metabolites in EVs. (B) VIP scatter plot identified by PCA showing the top 15 lipid metabolites. A: PKM2^fl/fl-C-EVs, B: PKM2^fl/fl-Hcy-EVs, C: LckCrePKM2^fl/fl-C-EVs, D: LckCrePKM2^fl/fl-Hcy-EVs. (C) Heatmap of changes in the EV phospholipid-containing polyunsaturated fatty acid profile. The relative levels of phospholipids containing polyunsaturated fatty acid metabolites, normalized to EV protein levels, are presented in the right panel. EV protein levels were normalized to lysate total protein. RAW264.7 cells pretreated with Dynasore (20 μmol/L) were further treated with EVs derived from different sources for 24 h (D–I). n = 3–5. (D) Iron concentrations in RAW264.7 cells. (E) Flow cytometric analysis of BODIPY 581/591 C11 in RAW264.7 cells. Quantification of oxidized BODIPY-C11 (emission: 590 nm)/reduced BODIPY-C11 (emission: 510 nm) ratio. Quantification of lipid peroxidation (LPO) (F) and malondialdehyde (MDA) (G) in RAW264.7 cells after incubation with EVs for 24 h. Measuring the concentrations of the antioxidant GSH (H) and NADPH/NADP⁺ ratio (I) in RAW264.7 cells. Peritoneal macrophages pretreated with Dynasore (20 μmol/L) were then treated with EVs for an additional 48 h. (J) Representative images of crystal violet staining were captured at 48 h after incubation to indicate migrated peritoneal macrophages. Quantification of the migrated cells is shown in the right panel. Cells were counted from 5 random microscopic fields for each sample in 5 independent experiments. The data are presented as the mean ± SEM. (C) *p < 0.05, compared with PKM2^fl/fl-C-EVs. #p < 0.05, compared with LckCrePKM2^fl/fl-C-EVs. Δp<0.05, compared with PKM2^fl/fl-Hcy-EVs. (D–J) *p < 0.05, compared with PKM2^fl/fl-C. #p < 0.05, compared with PKM2^fl/fl-Hcy. The data were compared using one-way ANOVA followed by Tukey's multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)