Materials and Methods Ethics Statement All procedures on mice were performed in accordance with the United Kingdom Home Office regulations. Mice b3-integrin-deficient and wild-type Tie1-Cre mice used for analysis of pSico lentiviruses in vivo were generated by crossing b3- integrin-deficient [61] and wild-type mice with Tie1-Cre trans- genic mice ([62]; kindly provided by Prof. Fa¨ssler, Max Plank Institute of Biochemistry, Martinsried, Germany), both on a mixed C57BL6/129 background. b3-integrin-deficient and wild-type mice were also bred with the ImmortomouseH (Charles River Laboratories, MA, USA) on a C57BL6 background and were used for preparation of immortalised endothelial cell cultures employed in some of the in vitro assays. Inducible vascular endothelium- specific Rac1-deficient mice (Rac1 flox/flox PDGFB-iCreER) were generated by crossing Rac1 flox/flox mice on a mixed C57BL6/129 background [28] with PDGFB-iCreER transgenic mice on a mixed C57BL6/CBAF2 background [29]. Isolation of mouse endothelial cells Mouse lung endothelial cells (MLEC) were isolated as previously reported [63]. Briefly, minced lungs were digested in collagenase type 1 (Gibco) and passed trough a 70 mm pore size cell strainer (BD Falcon) and cell suspension plated onto tissue culture plates coated with 0.1% gelatin containing 10 mg/ml fibronectin (Sigma) and 30 mg/ml Purecol (Nutacon). When cells reached ,80% confluency, macrophages were removed from the culture by immunosorting using rat anti-mouse CD16/CD32 Fcc receptor antibodies (AbD Serotech) and with two subsequent sorts using rat anti-mouse ICAM-2 antibodies (AbD Serotech) endothelial cells were positively selected. Endothelial cell purity was 96–98% as assessed by performing flow cytometry for PECAM-1 (clone MEC 13.3, Pharmingen), Endoglin (Pharmingen) and ICAM-2 expres- sion. Acquisition of samples was performed on a FACS Calibur fluorescence activated cell sorter (BD Biosciences) and the data were analysed with CELLQUEST software (BD Biosciences). Cells were cultured routinely in a 50:50 mix of Hams F12 and D- MEM media supplemented with 20% FCS, 6 mM glutamine, 25 mg/ml endothelial mitogen (Biogenesis), 1 mg/ml heparin and antibiotics. Generation of pSico-Rac1 lentivirus The plasmid for Stable RNA interference conditional (pSico) was a kind gift from Dr. Tyler Jacks (Massachusetts Institute of Technology, MA, USA) [64]. The pSico-Rac1 lentiviral vector was produced by digesting the pSico construct with HpaI-XhoI restriction enzymes (Promega), inserting shRNA oligonucleotides to target the mouse Rac1 gene and religating using T4 DNA ligase (Promega). A pSico containing shRNA to target the firefly Luciferase gene (pSico-Con) was generated to serve as a non- specific control RNAi and a pSico containing shRNA to target the mouse VEGFR2/Flk-1 gene (pSico-Flk-1) was made to stably knockdown the expression of Flk-1, therefore serving as a positive control in angiogenesis assays. Oligonucleotides coding shRNA for mouse Rac1, mouse VEGFR2/Flk-1, b3-integrin [65] and firefly Luciferase were designed using the program PSICOLIGO- MAKER 1.5 (available on Jacks’ website). Luciferase oligos were blasted against the mouse genome to ensure that they do not recognise any sequence in the mouse DNA. All oligonucleotide sequences are available upon request. Chemically competent E. coli (One Shot Stbl3, Invitrogen) were transformed with ligated Rac1 and Tumor Angiogenesis DNA. Plasmids were purified (QIAprep kit, Quiagen), digested with SacII-NotI (Promega) and positive clones released a fragment approximately 50 bp larger than the fragment released by pSico empty vector. Positive clones were sequence verified (Equipment Park Laboratory, London Research Institute Cancer Research UK). Sequences were analysed using DNA Strider 1.4f1 software (CEA, France). Lentiviruses were generated essentially as described in the Virapower Lentiviral Expression System User Manual (Invitro- gen). Briefly, 3 mg of pSico-Rac1, pSico-Con or pSico-Flk-1 lentiviral vectors and 9 mg of ViraPower Packaging Mix (Invitro- gen) were cotransfected in 293FT cells (Invitrogen) by using Lipofectamine 2000 reagent (Invitrogen). Supernatants were collected 48–72 hr after transfection, centrifuged at 3000 rpm for 15 min, filtered through a 0.45 mm filter, and used to directly infect primary mouse lung endothelial cells. Overnight infection usually was sufficient to infect 50–65% of the cells as assessed by flow cytometry. Acquisition of samples was performed on a FACS Calibur fluorescence activated cell sorter (BD Biosciences) and the data were analysed with CELLQUEST software (BD Biosciences). GFP-positive endothelial cells were sorted 3–4 days after transfection using a FACSAria cell sorter and program. Sorted cells were used to assess efficiency of Cre-mediated RNAi in vitro by transfecting them with Cre expression-plasmid, pTURBO-Cre (a kind gift from Dr. David Stevenson, The Beatson Institute, UK). Transfection of cells was performed by electroporation using the Basic Nucleofector kit designed for primary mammalian endothe- lial cells (Amaxa GmbH) following the manufacturer’s instructions. One week after transfection with pTURBO-Cre, Rac1 and Flk-1 knockdown efficiencies were assessed by RT-PCR and Western blotting. For in vivo experiments, lentivirus were ultracentrifuged at 25000x g for 1.5 hr and stocks titrated. Titers ranged between 1.5–26106 i.u/ml. Rac1 siRNA To target mouse Rac1 gene we used siGENOME ON- TARGETplus SMARTpool reagent (Dharmacon). Primary and immorto MLEC and mouse aortic tissue (see below) were transfected with anti-mouse Rac1 siRNA using Oligofectamine reagent (Invitrogen) following the manufacturer’s instructions. The final concentration of oligonucleotides in the transfection mixture was 100 nM and gene expression was examined 48 hr (MLEC) or 5 days (aortic tissue) post-transfection by RT-PCR and Western Blot analysis. In all transfection experiments scrambled siRNA (Dharmacon)-transfected samples were used as controls. Semiquantitative RT-PCR We used Rac1 isoform-specific primers derived from cDNA mouse sequences and PCR conditions that have been described previously [37]. As controls of the Rac2 and Rac3 reactions, total RNA and subsequent cDNA synthesis from spleen and brain tissues were obtained. Actin RT-PCR was performed as a loading control. Western blot analysis Cells were grown to ,80% confluency and lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF), supplemented with protease inhibitor cocktail II (Calbiochem). For preparation of lysates from aortas, 12–15 aortic rings were homogenised in 50 ml RIPA buffer by using a pestle and microwaved for 10–15 sec. Homogenates were centrifuged at 10,000 rpm for 10 min at 4uC, to pellet insoluble material. Lysates were subjected to SDS-PAGE PLoS ONE | www.plosone.org 14 March 2010 | Volume 5 | Issue 3 | e9766