(Invitrogen). The cells were observed with a Fluoview FV4000 confocal fluorescence microscope (Olympus, Tokyo, Japan). Western blot analysis Western blot analysis was performed as described previously [9]. Briefly, after the poly-acrylamide gel electrophoresis and membrane transfer, the membranes were blocked for 1 hour at room temperature with Tris-HCl–buffered saline (TBS) containing 5% skim milk after the transfer, and then incubated for 1 hour at room temperature in TBS containing 0.05% Tween 20 (T-TBS), 2.5% skim milk, and one of the following antibodies: anti-human VASH1 mAb (4E12) diluted 1/500, anti-SIRT1 Ab diluted 1/ 500, anti-SOD2 Ab, diluted 1/500 diluted 1/500 or anti- a-actin antibody diluted 1/10,000. After the membranes had been washed 3 times with T-TBS, they incubated for 1 hour with horseradish peroxidase-conjugated protein G (Bio-Rad, Hercules, CA). They were then washed again 3 times with T-TBS, after which the blots were detected by an enhanced chemiluminescence method using an ECL Western blotting detection kit (Amersham). The results were visualized by using a LAS-4000 (Fuji Film). Determination of SIRT1 activity SIRT1 activity was measured by using a SIRT1 Fluorimetric Drug Discovery Kit (Biomol International, Plymouth Meeting, PA) according to the manufacturer’s instructions. Briefly, cell lysates were extracted from VASH1 overexpressing or knocked- down HUVECs, and their deacetylation activity toward a peptide VASH1 Enhances Stress Resistance of Endothelium comprising amino acids 379–382 of human p53 (Arg-His-Lys- Lys(epsilon-acetyl)) was measured by use of a SpectraMax M2e with excitation at 360 nm and emission at 460 nm (Molecular Devices, Tokyo, Japan) Chromatin immunoprecipitation (ChIP) assay Total cell lysates (20 mg) derived from MS1 cells were immunoprecipitated with anti-rabbit IgG or anti-HuR antibody (5 mg) at 4uC for 3 hours. After that, 4 times-diluted Therma-Max UPA ProteinA (Magnabeat iIncorporated, Chiba, Ichihara, Japan) was added, and incubation was carried out at room temperature for 15 min. The complexes were collected according to the manufacturer’s instructions. Total RNAs were extracted from the complexes, and RT-PCR analysis was performed as described above. Trypan blue exclusion assay Cells were incubated for 5 min in a solution of 0.2% trypan blue in PBS. More than 100 cells were counted in each field, and the percentage of non-viable cells was calculated. Detection of cellular reactive oxygen species (ROS) ROS was detected by using Oxiselect in vitro ROS/RNS assay kit (Cell Biolabs, San Diego, Ca) according to the manufacture’s protocol. Briefly, the cell lysates (1 mg) from each treated cells were incubated with DCF-DiQxyQ for 30 min at room temperature. The fluorescence was measured by the use of SpectraMax M2e Figure 1. Knockdown of VASH1 induces premature senescence and enhances stress-induced cell death of HUVECs. (A) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, RT-PCR and Western blotting for VASH1 were performed. (B) Phase- contrast photomicrographs (on the left: 48 hours after siRNA transfection) and SA beta-gal staining (on the right: 5 days after siRNA transfection) are shown. Scale bars are 250 microm. SA beta-gal-positive HUVECs were quantified, and the % senescent cells was calculated. Values are the ratio of SA beta-gal-positive cells to total cells, and are means and SDs of 3 wells. (*P,0.01, N = 3). (C) HUVECs were transfected with VASH1 or control siRNA. After a 24-hour incubation, Western blotting for VASH1, ATM and p-ATM was performed. (D) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, LC3 (red) was immunostained. Scale bars are 25 microm. (E) HUVECs were cultured in growth medium with 100 microM H2O2 including mouse IgG (control) or 10 microg/ml VASH-1 antibody (4E12) for 48 h. Trypan blue exclusion assay was performed. Blue- stained cells quantified, and the % of dead cells was calculated (*P,0.01, N = 3). All the studies were repeated at least 3 times to confirm the reproducibility. doi:10.1371/journal.pone.0046459.g001 PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46459