FIG. 2.

Notch4 inhibits angiogenesis in the chick CAM in vivo. The avian retroviral packaging cell line Q2bn was transfected with empty vector CK (Q2bn-CK) or CK-Notch4IC (Q2bn-Notch4IC). On day 9, transfected Q2bn cell lines were placed onto nylon meshes on the CAM surface in the presence of VEGF (30 ng/ml). The grafted cells distribute throughout the mesh and secrete control virus or virus containing Notch4IC. Control CAMs were treated with medium alone or medium supplemented with VEGF. Images of the CAMs were captured on day 12. (A) CAMs treated with Q2bn-CK or Q2bn-Notch4IC cell lines in the presence of VEGF. Arrows, edges of the nylon mesh. Bars, 1 mm. (B) Vascular density quantitated after 4 days of incubation by counting the number of vessels that entered the mesh area and dividing by the perimeter of the mesh (vessels per millimeter). Data are the means ± standard errors from three experiments each done in replicates of four to six eggs. (C) Expression of HA-tagged Notch4IC in Q2bn cell lines verified by immunoblotting total cellular extracts with the anti-HA monoclonal antibody.