EGFL7 functions as an antagonist of Notch in HUVECs. (A) Human EGFL7 expression in lentivirally generated HUVEC lines, as measured by quantitative RT-PCR. Empty lentiviruses pCCL GFP and pLKO served as controls for EGFL7 overexpression and knockdown, respectively. Data are represented as fold induction relative to the lentiviral controls. (B) Proliferation assay of HUVEC lines grown in complete medium for 4 days. (C) Capillary sprouting assay. Control (GFP and pLKO), EGFL7 overexpressing (EGFL7), and knockdown (pLKO 61) HUVEC lines were coated on a cytodex bead, beads embedded in fibrin gels, and visualized on day 7. (D) HUVEC monolayer wounding assay at 0 and 24 hours. Dotted lines highlight the edges of the monolayer. (E) Quantitation of HUVEC migration in monolayer wounding assay, represented as percentage of area filled at 8 hours. (F) Transactivation of Notch/CSL-luciferase reporter in EGFL7 overexpressing and knockdown HUVEC lines. Data represented as relative luciferase units (RLU). (G) Notch4-dependent HUVEC morphogenesis assay. Control HUVECs (pCCL-GFP or pLKO) or EGFL7 overexpressing (EGFL7) or knockdown (pLKO61) cells were grown as monolayer on a fibrin gel. GFP- or Notch4/GFP-expressing HUVECs were overlaid on top of the monolayer. At day 7, cocultures were visualized and the number of GFP⁺ cells undergoing morphogenesis, as seen by the extending of processes into the surrounding matrix per field, was determined. Experiments were performed in triplicate, and error bars represent SD.