Expression of Y477F mutant ezrin abrogates tumour-induced angio/lymphangiogenesis. (A) Aortic rings were stimulated with CM from AC2M2 cells expressing pCB6 empty vector or Y477F ezrin and endothelial sprouting imaged (96 hr) and analyzed by ImagePro software (n = 3, bar graph). (B) Transwell migration of hLEC co-cultured with AC2M2 or Y477F cells (6 hr) was assessed by automatic count (ImageJ) of cells stained with DAPI. (C) Syngeneic Matrigel plug assay of AC2M2 or Y477F cells in CBA/J mice photographed on day 12 post injection along with representative H&E sections (arrows point to microvessels). Angiogenic activity was analyzed by MVD and vessel cross-sectional area (graphs to the right). (D) Lyve-1-positive cells (arrows) in plugs were quantified in selected hot spots by pathologist blinded to the study. P values were obtained from unpaired t test statistical analysis. Scale bars = 50 μm. CM: conditioned media; H&E: hematoxylin and eosin; MVD: microvessel density.