sEphB4 inhibits tubule formation on Matrigel in vitro. (A) HUVECs were cultured on standard Matrigel in growth factor-stimulated conditions with either sEphB4 or CF2 proteins for 24 hours. Shown are representative pictures from triplicate wells repeated twice. (B) Dose response. HUVECs were cultured as described in panel A with sEphB4 at indicated concentrations. (C) Quantitative analysis of data from panel B for tube length and the number of junctions in sEphB4-treated HUVECs (Bioquant Image Analysis; mean from triplicate wells in 2 repetition experiments). (D) sEphB4 inhibits invasion of HUVECs (top right) compared to cells treated with CF2 (bottom left) or no treatment (top left). Data are presented as number of invading cells ± SE from duplicate wells in 2 experiments (bottom right). (E) sEphB4 inhibits PDGF-stimulated attachment of HUAECs to Matrigel over 5 minutes and 15 minutes. Shown are number of attached cells as a percentage of control in triplicate wells; experiment was repeated twice. Photomicrographs in panels A, B, and D were taken using a Nikon Coolpix 5000 camera (Nikon, Tokyo, Japan) and a Carl Zeiss Invertoskop microscope (Zeiss, Goettingen, Germany) with a 4 ×/0.12 NA objective and 10 × eyepiece.