then stained with a 0.1% crystal violet solution. After extensive washes with deionized water, the dye was solubilized in 10% acetic acid solution, and the absorbance was measured at 595 nm. Cell migration assays HUVEC migration was evaluated by means of chemotaxis experiments in a 48-well modified Boyden chamber, as previously described [13,14]. Briefly, the filters coated with 10 mg/ml of type IV collagen were placed over a bottom chamber containing 25 ng/ml VEGF as attractant factor. The cells, suspended in 199 media containing 2% FBS, were added to the upper chamber at a density of 5.06104 cells/well. After 6 h of incubation at 37uC, the cells that had migrated to the lower side of the filter were stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, NJ, USA), and 5 unit fields per filter were counted by a scorer blind to the experimental conditions using a Zeiss microscope. The assays were run in triplicate. Determination of cGMP accumulation HUVECs were cultured in 60-mm Petri dishes and treated for 48 h with L-NAME or ODQ. During the last 30 min of treatment, 1 mM isobutylmethylxanthine (IBMX) was added to inhibit phosphodiesterases. cGMP was extracted in 500 ml of 0.1 N HCl, and its quantification was performed by an enzyme immunoassay (EIA) kit (Enzo Life Sciences, Vinci-Biochem, Vinci, Firenze, Italy) following manufacturer’s instructions for the acetylated assay procedure. Immunoblot and immunoprecipitation analyses For immunoblot analysis, HUVECs, plated in 35-mm diameter Petri dishes, were washed with phosphate-buffered saline (PBS), and then directly lysed in SDS-PAGE sample buffer (62 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.04% bromophenol blue). After SDS-PAGE electrophoresis, proteins were transferred onto nitrocellulose membranes that were blocked with 5% (w/v) non fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), and probed overnight with the indicated primary antibodies. After incubation with the appropriate peroxidase-conjugated secondary antibody (DAKO, Denmark), the immunoreactive bands were visualized by chemiolumines- cence (LiteAblot Plus, EuroClone, Italy). For KDR immunopre- cipitation, HUVECs were washed with PBS and lysed for 10 min on ice with RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM b-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate) supplemented with protease inhibitors. Aliquots of cleared cell lysates containing the same amount of protein (250 mg/sample) were incubated with anti KDR antibodies, followed by incubation with Protein A Sepharose. The immune complexes were washed with lysis buffer, eluted by boiling in sample buffer, and analyzed by SDS- PAGE. Densitometric analyses of the immunoblots were performed using the National Institute of Health (NIH) Image J program. ELISA determination of VEGF levels Cell supernatants were collected from HUVECs plated in 35- mm Petri dishes, and VEGF measurements were performed using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) following manufacturer’s instructions. VEGF levels were expressed relative to total cell protein (pg/mg of total protein) evaluated by BCA protein assay. NO Deficiency and HIF-1a6 Stabilization in HUVECs Preparation of nuclear extracts HUVECs, plated in 100-mm diameter Petri dishes, were washed with PBS and collected by scraping. The cells were then lysed for 10 min at 4uC in buffer A (10 mM HEPES pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.05% Nonidet P-40, 1 mM sodium orthovanadate) supplement- ed with protease inhibitors. After a 10 min centrifugation at 2,500 g at 4uC, the crude nuclei were washed with buffer A prior to lysis in buffer C (20 mM HEPES pH 8.0, 1.5 mM MgCl2, 420 mM NaCl, 1.0 mM DTT, 0.2 mM EDTA, 1 mM sodium orthovanadate, supplemented with protease inhibitors) for 30 min at 4uC. The nuclear extracts were clarified by centrifugation, and loaded on a 10% SDS-PAGE. The immuno- reactive bands were visualized by chemioluminescence after overnight incubation with the anti HIF-1a antibody at a 1:500 dilution in 5% BSA in TBS-T. Total RNA extraction for reverse transcription and quantitative real time PCR Total RNA was extracted using the RNeasyH Mini Kit and accompanying QIAshredderTM (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To avoid DNA contamination of samples, a 15 min on column incubation was carried out with DNase I (Qiagen). Reverse transcription was performed using the SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen), again following the manufac- turer’s instructions. For quantitative analysis of gene expression we used the ABI PrismH 7000 Sequence Detection System, SDS software version 1.2.3 (Applied Biosystems, CA, USA). Target sequences were amplified from 50 ng of cDNA. TaqManH Primer and Probe assays used were: human VEGF-A (Hs99999070_m1), KDR (Hs00176676_m1), eNOS (NOS3, Hs00167166_m1), HIF- 1A (Hs00153153_m1) and the endogenous control 18S (Hs99999901_s1). For calculation of results, the 22DDCt method was used allowing normalization to 18S and to the calibrator which is set to a value of 1. Transient transfection HUVECs, plated in 35-mm Petri dishes, were transfected with the expression vector pcDNA3ARNTdelta_b (DARNT) (kindly provided by Dr. Tacchini, Department of Human Morphology and Biomedical Sciences ‘Citta` Studi’, University of Milan, Milano, Italy), coding for a dominant negative mutant form of the HIF-1b ARNT subunit, and the void vector pcDNA3 using TransITTM LT1 (Mirus, Bologna, Italy). Six hours after transfection, the culture medium was replaced by fresh medium, and the cells were exposed to L-NAME for the following 48 h. Small interfering RNA (siRNA) transfection Validated StealthTM RNAi duplexes against human eNOS (GC content 48%) were provided from Invitrogen. As control RNA, we utilized a StealthTM RNAi negative control duplex (Medium GC Duplex, Invitrogen) with a 48% GC content, suitable for use as a control with StealthTM RNAi duplexes containing 45-55% of GC. All sets of RNAi molecules were transfected individually into HUVECs at a 30 nM concentration using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). The ability of the RNAi molecules to knockdown eNOS expression was analyzed 48 h after transfection by western blot analysis. Statistical procedures All data were expressed as mean 6 s.e.m. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29680