Notch1-Dll4 inhibition blocks sprouting and EphrinB2 expression in lymphatic vessels. (A) LYVE1 staining of the inner lymphatic network of a P7 ear. Box represents the area used for quantification of lymphatic branching. Scale bar represents 500 μm. (B) LYVE1 staining of the ear in control and αDll4 (10 mg/kg on P2/4) treated pups at P7. Arrows indicate sites of lymphatic sprouting. Scale bar represents 200 μm. (C) Quantification of the number of lymphatic branch points in PBS and αDll4 neonates (n = 5; P < .05). (D) LECs transwell migration assay toward VEGFC (300 ng/mL). Cells were either untreated (UT) or pretreated for 48 hours with 100nM DBZ before transwell assay. VEGFR3-Fc (10 μg/mL, VR3Fc) effectively inhibition of directed LEC migration toward VEGFC (n = 4; *P < .05). (E) qPCR analysis for Hey2 and EphrinB2 expression in lymphatic endothelial cells and HUVECs stimulated with human IgG or hDll4-Fc for 3 hours. (F) qPCR analysis for EphrinB2 and VEGFR3 expression in lymphatic endothelial cells treated with 100nM DBZ or transfected with siRNA targeting Dll4 or Notch1 (n = 3; *P < .05). (G) VEGFR3 and EphrinB2 costaining in P7 tail dermis isolated from neonates treated with PBS (control) or αDll4 (10 mg/kg on P2/5). Scale bar represents 10 μm. (H) Quantification of VEGFR3 and EphrinB2 expression in control and αDll4 treated pups (n = 3; *P < .01).