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. 2021 Apr 22;13(9):2009. doi: 10.3390/cancers13092009

Figure 5.

Figure 5

Downstream effects of cancer EVs on angiogenesis. (A) Tube formation assay of HUVECs incubated in basal medium (control), treated with 50 µg/mL MDA231-HEVs, or treated with both MDA231-HEVs and 0.1 µM Axitinib (inhibitor of VEGFR) for 24 h. The EVs induced formation of denser tube networks, which can be efficaciously blocked by Axitinib. Images are representative of two independent experiments. (B) The total tube length of images from the same experiment were analyzed by ImageJ and normalized by the average of the control group (N = 6). (C) MDA231-HEVs upregulated the VEGF-VEGFR angiogenesis pathway related gene, while showing no effect on the other ANGPT-TIE angiogenesis pathway in HUVECs (N = 3). (D) Western blot of proteins involved in the VEGF-VEGFR angiogenesis pathway. MDA231-HEVs conspicuously enhanced the level of VEGFR1 and the phosphorylation of downstream Erk1/2. Average intensities of the bands were first normalized to β-actin, and then normalized to the control group. Images are representative of two independent experiments. n.s.: not significant, * P < 0.05, ** P < 0.01 based on Mann–Whitney U test for (B), or Student’s t test for (C). Error bars stand for SEM for (B,C).