NO Deficiency and HIF-1a6 Stabilization in HUVECs Figure 6. Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP levels. (A) Characterization of HUVECs transfected with eNOS siRNA: densitometric analysis of eNOS protein expression where eNOS protein levels were normalized to b-actin protein. ***p,0.001; t test; n = 4. Inset: representative blots of eNOS protein in cells transfected with control (ctrl) or eNOS siRNA. (B) HUVECs were transfected with control (lane 2) or eNOS siRNA (lane 3), and HIF-1a protein was detected by western blotting on the corresponding nuclear extracts. In lane 1, nuclear extracts from untransfected cells. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies to check silencing, and with anti b-actin antibodies as loading control. A representative blot of 2 comparable experiments is shown. (C) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs 48 h after transfection with control or eNOS siRNA. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). *p,0.05; t test; n = 3. (D) MtDNA (left axis) and total cellular ATP content (right axis) were measured in HUVECs transfected for 48 h with control or eNOS siRNA. In silenced cells, mtDNA and ATP were reduced by 3660.4 and 4569.7% respectively. **p,0.01 and ***p,0.001; t test; n = 3. doi:10.1371/journal.pone.0029680.g006 methyl ester (L-NAME) in 199 medium containing 10% FBS for 48 h preceding the experiments. The concentration of L-NAME was chosen according to Papapetropoulos et al. [10]. PCR detection system (Bio-Rad) using 50 ng of total DNA. Calculations were performed with the 22DDCt methods using 18S rRNA as an internal control. Apoptosis assays Quantification of apoptosis/necrosis was performed by Annexin V-FITC conjugate and propidium iodide (PI) staining (Abcam, Cambridge, UK) followed by fluorescence activated cell sorting (FACS) performed with a FACScalibur flow cytometer equipped with a 488 nm argon laser (Becton Dickinson, San Jose´, CA, USA). The collected data were evaluated by Cell Quest software. The degree of apoptosis was calculated as apoptotic index considering cells both in early and late apoptosis. In addition, active caspase-3, Bcl-2 and Bax proteins were detected by western blot on total cell lysates. Evaluation of mitochondrial DNA (mtDNA) Total DNA was extracted with QIAamp DNA extraction kit (Qiagen, Hilden, Germany), and mtDNA levels were amplified with primers specific for the mitochondrial cytochrome b (CytB) gene and normalized to genomic DNA by amplification of the rRNA 18S nuclear gene. Primers used were: for CytB, F, 59– CTTCGCTTTCCACTTCATCTTACC-39 and R, 59-TTGGGTTGTTTGATCCT- GTTTCG-39, and for 18S, F, 59-CTGCCCTATCAACTTTCGATGGTAG-39 and R, 59-CCGTTTCTCAGGCTCCCTCTC-39. Quantitative real time PCR (RT-qPCR) reactions were run with the iQ SybrGreenI SuperMix (Bio-Rad, Segrate, Italy) on an iCycler iQ Real-Time Oxygen consumption Cellular oxygen consumption was measured as previously described [42]. Briefly, HUVECs were re-suspended in respiration buffer (0.3 M mannitol, 10 mM KCl, 5 mM MgCl2, 10 mM K2PO4, pH 7.4) [43] at a density of 3.06106/ml, and analyzed at 37uC in a gas-tight vessel equipped with a Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, UK) connected to a chart recorder. Protein content in cell samples was determined by the bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA). Cell metabolism assays Cell metabolism was assessed by means of a Cell Titer 96H Aqueous ONE Solution Reagent colorimetric assay (MTS, Promega, Madison, WI, USA), and the total cellular ATP content using a CellTiter-GloH Luminescent Assay (Promega). Both the assays were performed according to the manufacturer’s instruc- tions on HUVECs plated at a density of 26104 cells/well in 96- well microplates. Optical density at 490 nm (for MTS) and luminescence (for ATP) were measured using a multiplate spectrophotometer (VictorTM, PerkinElmer, Waltham, MA, USA). On parallel wells, cell proliferation was evaluated by crystal violet staining. Briefly, cells were fixed with 100% methanol, and PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29680