Fig. 5.

Extracellular vesicles derived from T lymphocytes promote iron accumulation in macrophages via PKM2.

Equal concentrations (10 μg/mL) of EVs from the PKM2^fl/fl-C, PKM2^fl/fl-Hcy, LckCrePKM2^fl/fl-C or LckCrePKM2^fl/fl-Hcy T lymphocytes cultured with RAW264.7 cells (A, C-D) or peritoneal macrophages (B) for 24 h n = 3–5. (A) Measurement of intracellular iron concentrations in RAW264.7 cells. (B) Free iron levels in peritoneal macrophages were measured by a Phen Green SK probe. As Phen Green fluorescence is quenched by iron, the inverse of fluorescence was plotted for these dyes to represent relative levels of cellular iron. (C) Protein expression and quantification (relative to β-actin) of Slc40a1 were measured via Western blots of RAW264.7 cells after EV treatment for 48 h. (D) Gene expression levels of Slc40a1 and Tfrc were measured via qPCR in RAW264.7 cells at 24 h after treatment with different EVs. RAW264.7 cells pretreated with the iron chelating agent deferoxamine mesylate (DFOM) (10 μmol/L) were further treated with different EVs. Measurements of the intracellular lipid peroxidation levels were made by quantifying oxidized BODIPY-C11 (emission: 590 nm)/reduced BODIPY-C11 (emission: 510 nm) ratio through flow cytometry (E), lipid peroxidation (LPO) analysis (F), and malondialdehyde (MDA) analysis (G) in RAW264.7 cells. Peritoneal macrophages pretreated with DFOM (10 μmol/L) were then treated with various EVs. (H) Representative images of crystal violet staining were captured at 48 h after incubation and quantification of migrated cells. Cells were counted from 5 random microscope fields for each sample in 5 independent experiments. The data are presented as the mean ± SEM. (A–D) *p < 0.05, compared with PKM2^fl/fl-C. #p < 0.05, compared with LckCrePKM2^fl/fl-C. Δp<0.05, compared with PKM2^fl/fl-Hcy. (E–H) *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.)