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Fig. 6 | Journal of Neuroinflammation

Fig. 6

From: Glutaminase 1 regulates the release of extracellular vesicles during neuroinflammation through key metabolic intermediate alpha-ketoglutarate

Fig. 6

EV release in immune-activated microglia is associated with the level of α-ketoglutarate. BV2 cells were treated with 10 μM BPTES or 10 μM CB839 4 h prior to LPS treatment overnight. BV2 cell regular medium was changed to serum-free DMEM medium when GLS1 inhibitors were added. Two hours prior to LPS treatment, 1 mM of α-KG was added to BV2 cells. a Protein lysates were collected from LPS-treated BV2 cells with or without GLS1 inhibitors or α-KG. GLS1 activities were determined by the enzyme activity assay. b Protein lysates were prepared from EV pellets. The levels of EVs markers Alix and flotillin-2 in EVs, as well as the levels of β-actin in whole cells, were determined by Western blot. EV protein loading was normalized with protein concentrations in the whole cell lysates. c, d Densitometric quantifications of the protein levels of in EV markers were presented as fold change relative to that in mock-infected control EV lysates. ANOVA and Bonferroni post-test; * and ** denote p < 0.05 and 0.01, respectively, compared with that of the control microglia cells, n = 3 per group. e After 1 mM of α-KG treatment, NTA was conducted with 100× dilution of collected EVs with filtered PBS. f Quantifications of the NanoSight NTA of vesicle concentration for samples from LPS-treated BV2 cell with GLS1 inhibitors and α-KG. ANOVA and Bonferroni post-test; * denotes p < 0.05 compared with that of the LPS-treated BV2 group. # denotes p < 0.05 compared with the corresponding GLS1 inhibitor group without α-KG, n = 5 per group

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