ZEB1 increases VEGFs secretion to induce tumor angiogenesis in vitro.
(A) MDA-MB-231 cells were transiently transfected with human ZEB1 expression plasmid or empty vector control. At the indicated time points, expression of ZEB1, VEGFA and VEGFC was verified by Western blotting. Actin was used as a loading control. (B) Production of VEGFA and VEGFC protein were verified by ELISA at the indicated time points following ZEB1 overexpression. *P < 0.05, **P < 0.01 vs respective control in one-way ANOVA followed by Tukey’s Honestly Significant Difference test. (C) HUVECs cultured in the presence or absence of VEGFA (20 and 40 ng/mL) or anti-VEGFA neutralized Ab (1 μg/mL) along with ZEB1/231-derived conditioned medium were subjected to a tube formation assay and photographed. (D) Quantification of tube formation was expressed as length of capillary tubes formed per mm2. *P < 0.05, **P < 0.01 vs respective control in Student’s t-test.