purified by glutathione-Sepharose 4B beads according to the manufacturer’s instructions (Promega). Cell Culture and Transfection Human umbilical vein endothelial cells (HUVECs, #CRL- 1730), HeLa (#CCL-2), and 293T (#CRL-11268) cells were obtained from the American Type Culture Collection. The use of HUVECs, HeLa, and 293T cells was approved by the Ethics Committee of Nankai University. HeLa and 293T cells in Dulbecco’s modified Eagle’s medium and HUVECs in RPMI 1640 medium were cultured in the indicated media supplemented with 10% fetal bovine serum at 37uC in a humidified atmosphere with 5% CO2. Plasmids were transfected into HeLa and 293T cells using polyethylenimine and transfected into HUVECs by electroporation with the ECM830 system (BTX). siRNAs were synthesized by Dharmacon and transfected with the RNAiMAX reagent (Invitrogen). In Vitro Angiogenesis Assays To examine the formation of tube-like structures, HUVECs were seeded onto 6-well plates precoated with matrigel, and tube formation was then examined at different time points. The extent of tube formation was quantified by measuring the cumulative tube length with ImageJ (National Institutes of Health). To examine capillary sprouting, HUVECs were suspended in culture medium containing 0.25% carboxymethylcellulose and seeded onto round-bottom 96-well plates to form spheroids. The spheroids were then embedded into collagen gels in 24-well plates, and culture medium was added on top of the gel. Capillary-like sprout formation was then examined by microscopy and angiogenic activity was quantified by measuring the cumulative sprout length per spheroid. Cell Migration Assays To examine wound healing, HUVECs grown in 24-well plates as conuent monolayers were mechanically scratched using a pipette tip to create the wound. Cells were washed with phosphate- buffered saline to remove the debris, and complete culture medium was added to allow for wound healing. Phase contrast images of the wound were then taken at different time points as described previously [23]. To analyze the transwell migratory activity of HUVECs, the upper surface of the transwell filters was coated with matrigel. Cells suspended in 200 ml serum-free media were then added to the chamber, and the chamber was placed in a 24-well plate containing complete medium. After 24-hour incubation at 37uC, the filters were gently taken out and matrigel on the upper surface of the filters was removed by cotton swabs. Cells on the underside of transwell filters were fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 10 minutes, and then photographed. The extent of cell migration was quantified as the number of migrated cells in the drug-treatment group divided by the number of migrated cells in the control group as described [24]. Cell Proliferation and Apoptosis Assays Sulforhodamine B staining assay was used to examine cell proliferation. In brief, cells were stained with 0.4% sulforhodamine B (Sigma-Aldrich) dissolved in 1% acetic acid. The cells were then washed with 1% acetic acid to remove unbound dye. The protein- bound dye was extracted with 10 mM Tris base to determine the optical density at 490-nm wavelength as described previously [25– 27]. Annexin V staining assay was performed to analyze the apoptotic rate. Cells were stained with fluorescein-conjugated HOP Regulates Cell Migration annexin V (Invitrogen), and phase contrast and fluorescence images were then taken with an Axio Observer A1 fluorescence microscope (Carl Zeiss) as described previously [28]. Examination of Cell Polarization HUVECs transfected with HOP or control siRNAs were scratched, and cells were fixed 2 hours later and stained with anti-a-tubulin antibody, anti-c-tubulin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue), respectively. To analyze membrane ruffle dynamics at the leading edge of migrating cells, cells transfected with the pEGFPC1 vector were cultured in a 37uC chamber on a TCS SP5 confocal microscope (Leica), equipped with a live-cell imaging workstation. The uorescence of GFP at the leading edge of cells was recorded at 20-second intervals using the LASAF software. The acquired image sequences were analyzed by ImageJ and membrane rufe dynamics were presented as three-dimensional surface plots. Immunofluorescence Microscopy Cells grown on glass coverslips were fixed with 4% para- formaldehyde for 30 minutes at room temperature and blocked with 2% bovine serine albumin in phosphate-buffered saline. Cells were incubated with primary antibodies and then with fluorescein or rhodamine conjugated secondary antibodies followed by staining with DAPI as described previously [29–31]. Coverslips were then examined with an Axio Observer A1 fluorescence microscope (Carl Zeiss). Immuoprecipitation and GST-Pulldown For immunoprecipitation, cell lysate was incubated with anti- GFP antibody-conjugated agarose at 4uC for 2 hours, and the precipitated proteins were then examined by immunoblotting. For GST-pulldown, cell lysate was incubated with bacterially purified GST or GST-HOP proteins immobilized on glutathione-conju- gated agarose at 4uC for 2 hours. The pulldown preparations were then analyzed by immunoblotting. Immunoblotting Cells were lysed in a buffer containing 1% Triton X-100, 150 mM NaCl, and 50 mM Tris (pH 7.5). Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked in Tris- buffered saline containing 0.2% Tween 20 and 5% fat-free dry milk and incubated with primary antibody and then with horseradish peroxidase-conjugated secondary antibody as de- scribed [32,33]. Specific proteins were visualized with enhanced chemiluminescence detection reagent (Millipore). Results HOP Promotes Endothelial Tube Formation and Sprouting To investigate the possible involvement of HOP in angiogenesis, we performed tube formation assay. HUVECs were transfected with HOP or control siRNAs and plated onto matrigel. Four hours later, cells transfected with control siRNA could form tube-like structures and 16 hours later, the tube-like structures became more significant. In contrast, knockdown of HOP expression apparently inhibited tube formation (Figure 1A–1C). We also found that overexpression of HOP could enhance the ability of HUVECs to form tubes (Figure 1D and 1E). In addition, we found that HOP shRNA could abolish the action of wild-type HOP in endothelial tube formation, but not the action of shRNA-resistant PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36389