Ezrin KD inhibits tumour-induced angio/lymphangiogenesis in vivo . (A) Matrigel plugs containing MDASrc (Src) or MDASrc shEZR (Src/shEZR) were digitally photographed on day 12 post injection in Rag2γ mice. Tumour-induced angiogenesis was analyzed in Matrigel plugs H&E sections for (B) MVD per selected field (manual count by trained pathologist blinded to the experiment), and (C) vessel luminal area (dots represent the mean vascular area in each selected field in pixel2). (D) Nature of blood vasculature was confirmed by CD31 immunostaining. (E and F) To assess tumour-induced lymphangiogenesis, Matrigel plugs were stained for lymphatic markers Lyve-1 and podoplanin followed by quantification of relative fluorescence density of each marker (bar graphs). (G) The Matrigel plugs derived from MDA231 (231) and MDA shEZR (shEZR) were harvested on day 20 post injection and assessed for MVD and vessel lumen area in corresponding H&E sections. (H) The effect of ezrin KD on MVD and vessel lumen size in tumours derived from MDA231 and MDASrc harvested on days 12 and 20 is summarized. Arrowheads in panels A and D point to microvessels in the tumour H&E sections. Inserts in panel A represent higher magnifications (×400) of areas in dotted boxes. P values were obtained from unpaired t test statistical analysis. All scale bars = 200 μm, except panel D bar = 50 μm. H&E: hematoxylin and eosin; KD: knockdown; MVD: microvessel density; Rag2γ: Rag2-/-II2rg-/- mice.