Fig. 2

EV release in HIV-1-infected macrophages is dependent on glutamine metabolism. MDM was infected by HIV-1 virus for 6 days, and the medium was changed into serum-free glutamine-free DMEM. Additional glutamine was added to the medium at the concentrations of 1, 2, and 5 mM. a, b Supernatants were collected and centrifuged at 1500 rpm for 5 min to remove cells. Samples were prepared for RP-HPLC, and the levels of glutamate and glutamine were determined. c, d Protein lysates were prepared from the whole cell lysates (c) and EVs pellets (d). The levels of EVs markers Alix and flotillin-2 in EVs, as well as the levels of β-actin in the whole cells, were determined by Western blot. EV protein loading was normalized with protein concentrations in the whole cell lysates. e, f Densitometric quantifications of the protein levels of EV markers were presented as fold changes relative to that in mock-infected control EV lysates. Western blot results shown are representative of the three independent experiments. Quantification results were normalized to the glutamine 5 mM group; ANOVA and post-test were performed on the remaining groups. *, **, and *** denote p < 0.05, 0.01, and 0.001, respectively, compared with that of the control microglia cells, n = 3 per group.