Fig. 1

KGA and GAC overexpression increase EV release in vitro. a Cell-free supernatants from control (CTL), Ad-GFP-, Ad-KGA-, and Ad-GAC-infected HeLa cells were incubated with or without 5 mM glutamine (Gln) and 1 mM L-DON ex vivo for 2 days. The glutamate levels were determined by RP-HPLC. b EVs were isolated from cell-free supernatants from four different groups of HeLa cells and incubated with or without 5 mM glutamine (Gln) and 1 mM L-DON ex vivo for 2 days. Glutamate production from EVs was determined by RP-HPLC. c Protein lysates were prepared from the whole cell lysates and EV pellets. The levels of EVs markers, tTG and flotillin-2, were analyzed by Western blot. EV protein loading was normalized with protein concentrations in whole cell lysates. Mitochondrial marker, cytochrome c, was used as a control to exclude contamination of EV pellets. d, e Densitometric quantifications of the protein levels in EVs were presented as fold change relative to that in control EV lysate. The protein levels in the whole cell lysates were presented as fold change relative to β-actin in the whole cells. Western blot results shown are representative of three independent experiments. CTL, control. Quantification results were normalized to the control group; ANOVA and post-test were performed on the remaining groups. *** and **** denote p < 0.001 and 0.0001, respectively, compared with the Ad-GFP group. ^### and ^#### denote p < 0.001 and 0.0001, respectively, compared with the 5 mM Gln group, n = 3 per group