Micrographs of zebrafish embryos were taken on a Stemi 2000 stereo microscope from Zeiss equipped with a DP200 CMOS digital camera and using DpxView Pro EE EF software, both from Deltapix . Confocal fluorescence micrographs of zebrafish embryos were acquired using a Nikon A1R confocal unit mounted on a Ti2000 inverted microscope . The microscope was equipped with 46 and 106 objective lenses, and fluorescence was revealed using a 488 nm laser line . For imaging, zebrafish embryos were anesthetized using 0.1 mg ml ethyl 3 aminobenzoate methanesulfonate in 0.36Danieau?s solution. Cell cultures Mouse aortic endothelial cells and bovine aortic endothelial cells were kindly provided by Prof. M. Presta . The cells were grown in Dulbecco?s modified minimum essential medium supplemented with 10 mM Hepes and 10 fetal calf serum . Cell proliferation assays Cells were seeded in 48 well plates at 10,000 cells per cm2. After 16 h, the cells were incubated in fresh medium in the presence of different concentrations of the test compounds .
On day 5, cells were trypsinized and counted by a Coulter counter . The compound concentration that inhibits cell growth by 50 was calculated based on cell counts GW9662 kinase inhibitor in control cultures. Cell migration assay Wounds were created in confluent MAE cell monolayers with a 1.0 mm wide micropipette tip. Then, cells were incubated in fresh medium containing 10 FCS in the presence of the test compounds. After 8 h, the wounds were photographed, and endothelial cells invading the wound were quantified by computerized analysis of the digitalized images. Tube formation assay Wells of a 96 well plate were coated with 60 ml matrigel at 4 uC. After gelatinization at 37 uC during 30 min, BAEC were seeded on top of the matrigel in 200 ml DMEM containing 1 FCS and the test compounds. After 6 hours of incubation, the cell structures were photographed at 1006magnification. Tube formation was quantified by counting the number of branching points.
Chorioallantoic membrane assay The in vivo CAM angiogenesis model was performed as described with slight modifications . Fertilized chicken eggs were incubated for 3 days at 37 uC when 3 ml of albumen was removed and a window was opened on the eggshell Romidepsin exposing the CAM. The window was covered with cellophane tape and the eggs were returned to the incubator until day 9 when the compounds were applied. The compounds were placed on sterile plastic discs , which were allowed to dry under sterile conditions. A solution of cortisone acetate was added to all discs in order to prevent an inflammatory response. A loaded and dried control disc was placed on the CAM approximately 1 cm away from the disc containing the test compound .