Fig. 1
Effects of different concentrations of EGFL6 protein on human umbilical cord vein endothelial cell (HUVEC) angiogenesis in vitro. a Phase-contrast images of HUVEC cultures treated with EGFL6 showing cell migration in the scratch-wound assay at the indicated times. Vertical dashed lines (white) demarcate the border between the wavefront of migrating cells and scratched area that was initially void of cells. b Quantitation (mean ± SD) of cell proliferation in response to EGFL6 (CCK-8 assay). c Mean percentage of cells migrating as a function of EGFL6 concentration in the scratch-wound assay. d, e Crystal violet-stained HUVECs that migrated in the transwell assay. Optical density (OD) of staining is relative to untreated control cells (e). f Relative quantification of capillary-like structures formed by HUVECs cultured with EGFL6 in the tube-formation assay. Values are relative to control values. g Phase-contrast images of HUVECs cultured with EGFL6 in the tube-formation assay. h Expression levels of Hif1a, VEGF-A, CD31, and EMCN genes in HUVECs treated with EGFL6 for 1 day, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. i, j Quantitation of VEGF-A protein concentration in HUVECs treated with EGFL6 (200 ng/ml) for the indicated times. k, l Western blots of lysates from HUVECs treated with EGFL6. Blots were probed with antibodies against angiogenesis markers (Hif1a, VEGF-A, CD31, EMCN) and pathway markers (β-catenin, pβ-catenin, active β-catenin, and pGSK3β). GADPH is the loading control. Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; *p < 0.05; **p < 0.01; and ***p < 0.001. All immunoblots were cropped from the original here and in subsequent figures. Experimental HUVECs were treated with the indicated EGFL6 concentrations. Control and experimental conditions for all functional assays were the same, except controls lacked EGFL6. Histogram values are based on three replicated experiments, and error bars are SD here and in all subsequent figures. Scale bars for a, e, g, 250 μm