Fig. 1
EVs increase the motility and invasion of breast cancer cells. a MDA-MB231 and MCF10-DCIS.com cells were isolated by size-exclusion chromatography (SEC). Fractions 5–10 were combined and characterized by NTA and BCA assay (b) and western blot (c). Human EV positive control was purchased from System Biosciences, consisting of EVs purified using Exoquick (System Biosciences). To demonstrate the effects of glycosylation, SEC-purified human EVs were treated with N-glycosidase F and blotted for CD63 primary ab. The lower bands (~ 25 kDa) in Hsp70 and CD63 blots are likely non-specific. d MCF10-DCIS.com cells were seeded ± 5 × 108 EVs in 96-well plates coated with 0.5 mg/ml Matrigel, and phase contrast images were taken over 96 h using an IncuCyte instrument after wounding. e For the invasion assays, cells were plated as in d and covered with a 2-mg/ml Matrigel pad after wounding. Cell invasion was determined ± 5 × 108 EVs over 36 h using an IncuCyte instrument. Averages of at least 4 independent experiments, each with 4 to 5 replicate wells, are shown. Groups were compared using two-way ANOVA, and p values were adjusted for multiple repeated measures. Error bars represent the standard deviation of the mean