Notch pathway components, the soluble inhibitor of Notch and its effects on the downstream Notch targets in LECs. (A) Comparison of mRNA expression levels of Notch signaling molecules between cultured human BECs and LECs. (B) qRT-PCR analysis of DLL4, NOTCH1, NOTCH4, HES1, HEY1, and NRARP in LECs treated with VEGF (100 ng/mL) or VEGF-C (100 ng/mL) for 1 hour. The dashed line indicates the nontreated control levels, which are taken as 1. (C) Diagram of the domain structures of human and mouse full-length Dll4 and mDll4-Fc. The latter comprises the extracellular domain (ECD) of mDll4 and the Fc domain of human IgG. (D) Western blot analysis of cleaved N1ICD in LECs treated with Dll4-Fc or hIgG (10 μg/mL) overnight. β-actin served as a loading control. (E) Immunofluorescent staining for cleaved N1ICD (red), PECAM-1 (green), and DAPI (blue) of LECs treated with Dll4-Fc (10 μg/mL) overnight or untreated. Arrows indicate diminished N1ICD staining in the nuclei and arrowheads the active N1ICD. Scale bar, 50 μm. (F) Quantification of N1ICD in the nuclei in panel E. (G) qRT-PCR analysis showing inhibition of Notch target gene expression by Dll4-Fc in LECs 24 hours after treatment. (H) qPCR analysis of DLL4 expression in LECs treated with Dll4-Fc– or HSA-conditioned medium for 24 hours. Data represent means ± SEM of at least 3 independent experiments; *P < .05; **P < .01; ***P < .001.