Experimental Procedures Materials-Fetal bovine serum (FBS) and trypsin were obtained from Gibco (Carlsbad, CA, USA). Antibodies against paxillin, ERK, p38, JNK, Akt, GAPDH, cofilin, GFP, p110a, p110b and p110c were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against actin, p110d and HA were purchased from Santa Cruz Biotechnology (CA, USA). The antibody against paxillin that was used for immunofluorescence was purchased from Abcam (Cambridge, UK). Rho GTPases pull down activation assay kits and antibodies against Rac1, Cdc42, and RhoA were from Millipore (Temecula, CA, USA). All of the pharmacologic inhibitors such as LY 294002, U0126, NSC23766, and Su5416 were obtained from Tocrics (Bristol, UK). Growth factor reduced matrigel and human recombinant VEGF were from BD Biosciences (Bedford, MA, USA). Gelatin and NaHS were from Sigma (St Louis, MO, USA). Human recombinant Rac1 and G-LISA Rac1 activation assay kits were obtained from Cytoskeleton (Denver, CO, USA). Cell culture-Primary human umbilical vein endothelial cells (HUVECs) were obtained from AllCells (Emeryville, CA, USA). Each batch of HUVECs was examined using flow cytometry and was shown to be 99% CD31 positive, with a viability of 95%. Cells were cultured in complete HUVECs medium on plates coated with 0.5% gelatin. HUVECs (passage 3 or 4) that were approximately 70% confluent were used for most experiments. Cells were serum starved in 0.1% fetal bovine serum in endothelial cell basal medium for 24 h prior to the various treatments. Cells were passaged 1 day prior to electroporation to ensure that they were actively growing on the day of electroporation. Monolayer wound healing assay and transwell boyden chamber assay-For the monolayer wound healing assay, confluent HU- VECs were starved for 24 h before starting the experiments. The cell monolayer was wounded by scratching with a plastic pipette and was washed three times with PBS. Images were taken at 0 h or 6 h after wounding, and the wound closure was calculated 6 h later. The transwell migration assays were performed as previously described with little modification [18]. Briefly, cells (56104/well) were plated on 8 mm pore transwell filters (Corning, Lowell, MA, USA) that were coated with 0.5% gelatin. After 16 hours, the filters were fixed with 4% formaldehyde and stained with 0.4% crystal violet in 10% ethanol. Non-migrated cells on the upper side of the filters were gently removed using a cotton swab, and cells on the underside of the filters were photographed. To quantify cell motility, cells that stained positively with crystal violet in nine random fields of each filter were counted, and three independent filters were analyzed. In vitro endothelial tube formation assay-Growth factor- reduced matrigel matrix was plated evenly in a 24-well plate (200 ml/well) and incubated at 37uC for 30 min before adding HUVECs (75,000 cells/well). Then, 16 h after seeding, photo- graphs of representative 106 fields were taken (n$5 per condition and genotype), and endothelial tubes were quantified by counting branches and tube length. Plasmids-pcDNA3-EGFP-Rac1-T17N (Addgene plasmid 12982) as the dominant negative plasmid of human Rac1 gene and pcDNA3-EGFP (Addgene plasmid 13031) as the empty control vector were both obtained from the plasmid repository at Addgene (Cambridge, MA, USA). Hemagglutinin (HA)-tagged dominant-negative (DN) Akt (kinase-inactive mutant Myr-Akt- K179M) [24] was a kind gift from Dr. Jin Q. Cheng (Department of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine, H. Lee Moffitt Cancer Center, H2S Signals through VEGFR-PI3K-Rac1-Cofilin Tampa, Florida). All the plasmids were confirmed by sequencing before amplification. Since HUVECs are hard to transfect, electroporation as described below was used to transfect the cells. RNA interference-All small interfering RNAs (siRNAs) were synthesized by Ambion (Austin, TX, USA). Oligonucleotide sequences were as follows: Rac1 siRNA (sense: 59-GGAACUAAA- CUUGAUCUUAtt-39, antisense: 59-UAAGAUCAAGUUUA- GUUCCca-39), p110a siRNA (sense: 59-GUAAUUACCCA- GAUCCUAUtt-39, antisense: 59- AUAGGAUCUGGGUAAUUACag-39), p110b siRNA (sense: 59-GGGAAAGCUGGACUACUAAtt-39, antisense: 59-UUA- GUAGUCCAGCUUUCCCtg-39), p110c siRNA (sense: 59- GCUUUAGAGUUCCAUAUGAtt-39, antisense: 59- UCAUAUGGAACUCUAAAGCtt-39) and p110d siRNA (sense: 59-GACUAAUAAUAGUGAGAAAtt-39, antisense: 59-UUUCU- CACUAUUAUUAGUUCtt-39). For RNA interference experi- ments, HUVECs were electroporated with scrambled siRNA as a negative control (Ambion), GAPDH siRNA (Ambion) as a positive control and each of the targeting silencer oligonucleo- tides. Electroporation-Prior to electroporation, the cells were washed with PBS, counted, and resuspended in Gene Pulser electropora- tion buffer (Bio-Rad, Hercules, CA, USA) to a density of 16106 cells/ml, and mixed with nucleic acid. A fluorescently labeled transfection control siRNA (Ambion) was used to assess the electroporation efficiency and to screen for electroporation parameters. The plasmids were used at a concentration of 20 mg/ml, and silencer siRNAs were used at 100 nM. The cells and nucleic acid were gently mixed in Gene Pulser electroporation buffer, and 600 ml of the mix was transferred into 4 mm electroporation cuvette (Bio-Rad). Electroporation was carried out using a manually entered protocol (250 V, 10 ms) on the Gene Pulser Xcell electroporation system (Bio-Rad). The electroporated cells (200 ml) were transferred into a 6-well tissue culture plate containing 2 ml of growth media/well and were incubated at 37uC for 24 h, unless otherwise indicated. Analysis of transfection-Cells that were electroporated with EGFP-containing plasmids or fluorescent control siRNA were observed and photographed under a fluorescence microscope 48 h after electroporation. Delivery of silencer siRNA was also assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from cells using an RNArose reagent, and cDNA was synthesized using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Gene-specific primers were used to amplify relevant messages using SYBR Green Master Mix (Toyobo) on an iCycler iQTM real-time PCR detection system (Bio-Rad). All RT-qPCR reactions were carried out in triplicate. For the Rac1 gene, the forward primer was 59-GCCAATGT- TATGGTAGAT-39 and the reverse primer was 59-GACTCA- CAAGGGAAAAGC-39. For the GAPDH gene, the forward primer was 59-AACGGATTTGGTCGTATTG-39 and the re- verse primer was 59-GCTCCTGGAAGATGGTGAT-39. For the actin gene, the forward primer was 59-CACCAACTGGGACGA- CAT-39 and the reverse primer was 59-ACAGCCTGGATAG- CAACG-39. Immunofluorescence and confocal microscopy-HUVECs grown on coverslips were fixed by 4% paraformaldehyde in PBS for 30 min, permeabilized in 0.1% Triton X-100 and blocked in PBS containing 2% BSA for 1 h at room temperature. The samples were incubated with primary antibodies overnight at 4uC, followed by incubation with Alexa Fluor-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The cells were washed 3 times with PBS and stained for F-actin and DNA with Rhodamine-phalloidin (Invitrogen) and PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44590