G. Top: graphical representation of the functional (invasion) assay performed using MDA‑MB‑231 derived EVs on MCF7 recipient cells. Bottom: Boyden chamber invasion assay in 3D matrigel of EVs isolated from different steps of ultracentrifugation. EVs were collected from conditional medium produced by MDA-MB-231 expressing Control (pcDNA3) or FLAG-RASSF1C. The 2K (large EVs), 10K (medium EVs or MVs) and 100K (small EVs, including exosomes) pellets were collected and equal amount of EVs (normalized according to protein content) were incubated with MCF7 cells. MCF7 were evaluated for invasion after 24 hours. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. 