Mult VN promotes VE-cadherin internalization.

<p>(A) HUVECs were treated with mult VN (10 µg/mL). Confocal microscopy showed continuous and organized VE-cadherin staining, which is normally observed in ECs at cell-cell junctions (A-a). VN stimulation resulted in a disorganized pattern of VE-cadherin distribution at the membrane and led to intracellular accumulation of VE-cadherin (A-b); (B). To further determine whether cell surface-derived VE-cadherin was internalized into intracellular compartments, cells were prepared without acid washing and analyzed using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037195#s4" target="_blank"><i>Methods</i></a>. A time-dependent internalization of VE-cadherin was observed (upper panels, B-a-d). Pretreatment with LM 609 (LM) and su6656 (su) prevented VN-mediated VE-cadherin internalization (B-e, f, lower panels). VE-cadherin internalization was not observed after treatment with mono VN (10 µg/mL)(B-h). VEGF (50 ng/mL) as a positive control (B-g). (C). Cells were prepared by acid washing and analyzed using confocal microscopy and cell surface VE-antibody was removed with a low pH acid wash. VN treatment increased the intracellular accumulation of VE-cadherin internalized from the cell surface (C-b, c) relative to no treatment (C-a). Nuclei are stained with DAPI. The scale bars represent 10 µm. Arrows: cell surface; Arrowhead: intracellular accumulation. (D). HUVEC monolayers were treated with VN at the indicated dose. Cells were lysed using RIPA buffer for western blotting analysis of total VE-cadherin. (E). Cells were treated with trypsin/EDTA, pelleted and lysed with RIPA for western blotting analysis of intracellular VE-cadherin. (F). HUVECs were transfected with Src siRNA or scrambled siRNA (con siRNA). The efficiency of Src protein expression knock-down was analyzed using immunoblot (left panel). Cells were treated with trypsin/EDTA, pelleted and lysed with RIPA for western blotting analysis of intracellular VE-cadherin (right panel). β-actin was used as a loading control.