The ankyrin repeats are required for Notch4-mediated inhibition of endothelial sprouting. (A) Phase-contrast micrographs of microcarrier beads seeded with HMECs transduced with N4IC or empty vector control and stimulated with FGF-2. Arrows indicate capillary-like sprouts of sufficient length to be counted after 3 days of stimulation. Original magnification, × 100. (B) Transmission electron micrographs of sectioned fibrin gels containing sprouting HMECs. Panel i demonstrates the base of a sprout forming a lumen that excludes the fibrin gel (original magnification, × 9000; bar = 5 μm. Panel ii demonstrates another lumen formed by HMECs. Arrowheads point to adherens-like junctions and the arrow points to a coated pit (original magnification, × 54 000; bar = 1 μm. Electron microscopy was performed on a Philips 400 transmission electron microscope, and images were photographed with the built-in 3550 × objective (i) and the 21 500 × objective (ii). Images were scanned on an Epson Perfection scanner using Photoshop Element. (C) Quantitation of sprouting for the transduced cell lines after 3 days of stimulation with basal medium or medium supplemented with FGF-2 or VEGF. The number of sprouts per microcarrier bead (sprouts/bead) were counted and graphed as means + SD. Data are from a single experiment done in triplicate. The relative sprouting patterns are representative of at least 4 separate experiments.