Ezrin KD inhibits lymphangiogenic activity of breast cancer cells . (A) The expression levels of active Src (pY416), pT567 ezrin/pT558 moesin (pTERM) and total ezrin in MDA231, MDASrc (Y527F Src in pWZL vector) and MDASrc shEZR (shRNA in pLKO.1 vector) cells were validated by western blot. Over 85% of endogenous ezrin was depleted in our shEZR cells. (B) The relative rates of proliferation (normalized against 24 hr) of MDA231 (231), MDASrc (Src) and MDASrc shEZR (Src/shEZR) cells were assessed by an MTT assay. (C) hLEC migration assay: percent migration of hLEC, co-cultured with MDA231 (231), MDASrc (Src), or MDASrc shEZR (Src/shEZR) cells (6 hr), is presented following normalization against the MDA231-treated group. (D) Tube formation assay: hLEC were stimulated with CM from MDA231, MDASrc, and MDASrc shEZR for 4 hr prior to manual counting of the tube-like structures (marked by asterisks). The histogram displays the percentage of tube formation in each group relative to MDA231 control cells. Scale bar = 50 μm. (E) The integrity of the hLEC monolayer was assessed by the paracellular diffusion of TMR-dextran (2000 kDa). Readings are expressed in relative fluorescence as percentage of control (collagen-coated membrane alone) (n = 3). (F) Monolayer of hLECs (5 × 105 cells) was co-cultured overnight with no cells (control), MDASrc, or MDASrc shEZR (shEZR). The diffusion of TMR-dextran across the hLEC barrier was measured in 90 min and presented as relative fluorescence normalized to the control (n = 4). CM: conditioned media; hLEC: human lymphatic endothelial cells; KD: knockdown; kDa: kiloDaltons; shRNA: short hairpin RNA.