Figure 1.

Extracellular glutamate promotes EV release. (a and b) MDA-MB-231 cells were incubated for 48 h in glutamine-replete medium with or without the mGluR3 antagonist LY95 (0.3 µM) or vehicle control (Ctrl; a) or in glutamine-deficient medium in the presence or absence of Glu (20 µM; b). EVs were isolated and analyzed by nanoparticle tracking. Values were normalized to cell number and are expressed as the number of particles of a given size in 0.5-nm increments. The bar graphs show the relative number of particles released per cell with diameter <160 nm, corresponding to the size of EVs. Values represent mean of n = 4 independent experiments. Cell lysates and differential centrifugation pellets were also analyzed by Western blotting for the indicated EV markers. Actin was used as a loading control. (c) Isolated EVs from cells treated with or without LY95 were fixed and placed onto formvar-carbon–coated grids. Dark spots indicate nanogold labeling of CD63. Bar, 200 nm. Diameter of EVs is plotted as the number of a given size in 10-nm increments, and total number of EVs per field is shown on the righthand graph from 40 different fields. Dotted lines and error bars represent SEM. ***, P < 0.001; all statistical analyses by t test with Welch’s correction.