Fig. 3.

sEV-VEGF is signaling competent. a Immunoblot of phosphorylated VEGFR2 (p-VEGFR2) and total VEGFR2 in HUVEC at 5 min following stimulation with sEVs of ES2, HCT116, and 786-0 cells or with recombinant VEGF₁₆₅ (rVEGF₁₆₅). b–e HUVEC were pretreated with inhibitors of VEGFR tyrosine kinase activity (b, c) and with neutralizing Ab to VEGFR2 (d, e), stimulated with sEVs or rVEGF₁₆₅, and then assayed for tube formation at 4 h thereafter. In b and d, mean ± SD of n = 4 independent experiments. In c and e, representative images of tube formation. Scale bar = 100 μm. f VEGF levels in conditioned media (CM) of ES2 and HCT116 cells that were depleted of sEVs (sEV-dep) or left non-depleted (whole). Shown are mean ± SD of n = 3 independent experiments. g–i HUVEC were stimulated with whole and sEV-depleted conditioned media, and then assayed for VEGFR2 phosphorylation (g) and tube formation (h, i). In h, mean ± SD of n = 4 independent experiments. In i, representative images of tube formation. Scale bar = 100 μm. **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way ANOVA with Bonferroni’s corrections in b, d, and h, and by a two-sided unpaired t-test in f. Source data used for graphs in b, d, f, and h can be found in Supplementary Data 3