HUVEC Tubule Formation Assay These assays were performed as described in [68]. The constructs used are described in [32]. Mouse lung endothelial cell growth Temperature-sensitive immortalised wild-type and b3-null mouse lung endothelial cells were plated at density of 1,500 per square centimetre on pre-coated tissue culture plastic. siRNA transfections were performed and cells were allowed to continue growing. Cells were trypsinized from triplicate wells every day for up to three days and counted using a haemocytometer (Fast Read 102 cell counting chamber [ISL, Paignton, UK]). Cell viability was assayed by Trypan blue exclusion method. Wound closure scratch assays Wound closure scratch assays were performed as described (21). Briefly, immortalised b3-null and wild-type MLEC were plated at a density of 10,000 per square centimetre on pre-coated tissue culture plastic. siRNA transfections were performed and cells were grown until a confluent monolayer was achieved. Cells were serum starved for 3 hr, monolayers scratched with a P200 tip, and the scratches were photographed using an inverted microscope (Carl Zeiss Axioplan) equipped with a camera (Hamamatsu Photonics, Ltd.) at three points along their length. The medium was changed for 50:50 mix DMEM:Hams F12 plus glutamine supplemented with VEGF-A164 (25 ng/ml) and cells allowed to migrate over 8 hr at 37uC. Scratches were rephotographed, and the percentage of migration was determined relative to the scratch width at 0 hr. Modified Boyden chamber assays: 3D migration assay One day before setting up the 3D migration assays, Con- and Rac1- siRNA transfected b3-null and wild-type and immortalised MLEC were treated with 4 mg/ml Mitomycin C (Sigma, Dorset, UK) at 37uC for 2 hr, to avoid changes in cell proliferation. Cells were washed twice in PBS and serum starved overnight in D- MEM:Ham’s F12 plus L-glutamine. Matrix solution was prepared by mixing 1 part of GFR-Matrigel with 1 part of D-MEM:Ham’s F12 media. 70 ml of matrix solution was pipetted into the upper well of the transwell (Costar, MA, USA) and incubated at 37uC for at least 60 min. D-MEM:Ham’s F12 plus L-glutamine and 2.5% FCS supplemented with growth factor alone or growth factor and inhibitor (VEGF-A164 [25 ng/ml] and DC101 [50 ng/ml]) were pipetted into the ‘lower chamber’. Cells resuspended in D- MEM:Ham’s F12 plus L-glutamine and 2.5% FCS at 50000– References 1. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438: 932–936. 2. Silva R, D’Amico G, Hodivala-Dilke KM, Reynolds LE (2008) Integrins: the keys to unlocking angiogenesis. Arterioscler Thromb Vasc Biol 28: 1703–1713. 3. Eskens FA, Dumez H, Hoekstra R, Perschl A, Brindley C, et al. 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Cells at 120,000 cells/ml in DMEM:Hams F12 plus glutamine were plated per well/coverslip and incubated at 37uC. Every two hours cells were observed under an inverted microscope (Carl Zeiss Axioplan) equipped with a camera (Hamamatsu Photonics, Ltd.), and photographed. Branch points of developing tubular structures were counted and compared at 8 hr time-point. Analysis of Statistical Significance Data sets were analysed for significance using Student’s t test. P,0.05 was considered statistically significant. Experiments were carried out in a double blind fashion and all experiments were performed in triplicate. Acknowledgments We would like to thank Reinhart Fa¨essler (MaxPlank, Germany) for providing us with the Tie1-Cre mice; Tyler Jacks (MIT, USA) and David Stevenson (The Beatson Institute, UK) for the pSico and pTURBO-Cre constructs, respectively; Dietmar Vestweber (MaxPlank, Germany) for the endomucin antibody; John Collard (King’s College, UK) for the GST- PAK-CRIB construct and Vania Braga (Imperial College, UK) for technical advice with its use in pull-downs; and Ian Hart (Bart’s and The London, Queen Mary’s School of Medicine and Dentistry, London, UK) for his help in the preparation of this manuscript. Author Contributions Conceived and designed the experiments: GD SDR KHD. Performed the experiments: GD SDR MG LER GJT GS MF VLJT GM. Analyzed the data: GD SDR KHD. Contributed reagents/materials/analysis tools: GE. Wrote the paper: GD SDR KHD. 7. Nabors LB, Mikkelsen T, Rosenfeld SS, Hochberg F, Akella NS, et al. 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