<i>In vivo</i> and <i>in vitro</i> angiogenesis induced by SW480-derived EVs.

<p>(A, B) EVs released by SW480 cells were purified from culture supernatants by a combination of differential centrifugation, ultracentrifugation onto sucrose cushions, and iodixanol density gradients. Each fraction of iodixanol density gradients was analyzed by Western blotting to detect CD81 and CD63, marker proteins of EVs (A). The purified EVs in fraction 3 were analyzed by Western blotting to detect non-EV marker proteins (GM130 and cytochrome <i>c</i>). SW480-derived whole cell lysate (WCL; 10 µg) and SW480-derived EVs (EVs; 10 µg) were loaded for Western blotting analysis (B). (C, D) Matrigel in the presence or absence of SW480-derived EVs (20 µg) was injected subcutaneously into C57BL/6 mice. After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel C. Scale bars represent 100 µm. Fluorescence intensities of CD31 staining of the Z-stack plane of the Matrigel were measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115170#s2" target="_blank">Methods</a> (D). (E) Migratory activity of SW480-derived EVs was evaluated by a scratch wound-healing assay. Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (F) Proliferative activity of SW480-derived EVs (1 µg/mL) was evaluated by assessing the mitosis marker PH3. After 24 h, the PH3 and nuclei were stained with anti-PH3 antibody and Hoechst, respectively and evaluated by confocal microscopy. The percentage of PH3-positive cells in HMEC-1s was quantified by counting the cells with co-localized fluorescence signals (n = 3). As a positive control, HMEC-1s were treated with EGM-2 medium supplemented with diverse angiogenic factors such as EGF, FGF2, VEGF, and IGF1. Data are represented as mean ± SD. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; n.s., not significant.