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. 2020 Jun 5;9(6):1413. doi: 10.3390/cells9061413

Figure 2.

Figure 2

Δ21-121 EMCN is the minimal truncation mutant to rescue vascular endothelial growth factor (VEGF)-induced migration, tube formation, and cell proliferation of primary human endothelial cells. (A) HRECs were transduced with adenoviruses expressing the FL and different EMCN mutants individually, and endogenous hEMCN was knocked down using siRNA with non-targeting siRNA (siNT) as control. Then, VEGF-stimulated closure percentage was normalized to that without VEGF for each treatment group. EMCN knockdown abolished VEGF-induced migration, and Δ21-161EMCN failed to rescue, unlike other mutants. (B) Representative images of cell migration: The white line indicates the initial wound area (0 h), and the yellow line marks the wound area after 15 h. Plus and minus indicates the addition of VEGF. Scale bar: 100 um (C,D) Tube formation by HREC at 6 h was recorded, and the total tube length and total segment length were quantified using Image J angiogenesis plug-in in a masked fashion. The FL EMCN, Δ21-81, and Δ21-121 all rescued VEGF-induced tube formation whereas Δ21-161 did not. (E) Representative images for the tube formation assay. Scale bar: 400 µm. (F) HRECs were incubated in serum-free media with and without 10 ng/mL VEGF and cell number following 48 h of incubation quantified. The FL EMCN, Δ21-81, and Δ21-121 all rescued VEGF-induced HREC proliferation whereas Δ 21-161 did not. All data = mean ± SEM, ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by 2-tail unpaired t-test, n ≥ 3.