starvation-induced stress. The significance of this effect and the underlying mechanism is examined in this study. Materials and Methods All of the animal studies were reviewed and approved by the Center for Laboratory Animal Research, Tohoku University in accordance with established standards of humane handling of research animals. Materials The following materials and their sources were used: a-minimal essential medium (aMEM) and Dulbecco-modified Eagle medium (DMEM) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); Superscript One-step RT-PCR with platinum Taq, Lipofectamine RNAi max, Opti-MEM I, stealth siRNAs, and 5– 6-chloromethyl-29, 79-dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H2DCFDA) from Invitrogen (Carlsbad, CA); endothe- lial basal medium (EBM) and endothelial cell growth supplements from Clonetics (Walkersville, MD); Isogen from Nippon Gene (Toyama, Japan); Hybond-ECL from Amersham (Buckingham- shire, UK); N-acetylcysteine (NAC), SU5416, vascular endothelial growth factor (VEGF), protein G Sepharose, anti- b-actin antibody from Sigma (St. Louis, Mo); hydrogen peroxide from Mitsubishi Chemical Corporation (Tokyo, Japan); anti-8-hydro- xydeoxyguanosine (8-OHdG) antibody from Abcam (Cambridge, MA); anti-silent mating type information regulation 2 homolog 1 (SIRT1) antibody, anti-super oxide dismutase 2 (SOD2) antibody, anti-HuR antibody, ataxia teleangiectasia mutation (ATM) antibody, phospho-ATM antibody (Ser1981), anti-rabbit IgG and SIRT1 activator 3 from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-light chain 3 (LC3) antibody from Medical & Biological Laboratory (Nagoya, Japan). Horseradish peroxidase (HRP)-conjugated anti-human VASH1 mAb (4E12) was described previously [2]. Cells Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were obtained from Sanko Junyaku Industries (Tokyo, Japan) and were cultured on type I collagen-coated dishes (Iwaki, Chiba, Japan) in EBM containing endothelial cell growth supplements and 2% fetal bovine serum (FBS). All experiments using HUVECs and HAECs were performed at population doubling levels of less than 10. Normal human bronchial epithelial cells (NHBECs) were obtained from Lonza (Basel, Switzerland) and were cultured in BEGM Bullet Kit (Lonza). Mouse EC line MS1, a cell line immortalized from pancreatic ECs by SV40 large T antigen, were purchased from American Type Culture Collection (ATCC, Manassas, VA). The MS1 cells were cultured in aMEM supplemented with 10% FBS, as described previously [8]. VASH1 overexpression in HUVEC and MS1 VASH1 overexpression in human unblical vein endothelial cells (HUVECs) or in human aortic endothelial cells (HAECs) was achieved by infection with a non-proliferative adenovirus vector encoding human VASH1 (AdVASH1) at a final multiplicity of infection of 30 [2]. Alternatively, HUVECs were transiently transfected with the VASH1 expression plasmid vector [8]. VASH1 overexpressing MS1 stable clones were described previously [8]. VASH1 Enhances Stress Resistance of Endothelium Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNAs were extracted and RT-PCR was performed by using a One-step RT-PCR kit (Invitrogen) according to manufac- turer’s instructions. Primer pairs used in this study were as follows; human VASH1 forward, 59- ATG GAC CTG GCC AAG GAA AT-39,and reverse, 59- CAT CCT TCT TCC GGT CCT TG- 39;human NOX1 forward, 59-CGT CTG CTC TCT GCT TGA AT-39, and reverse, 59-TGA ATC CCT AAG CCA AGG AT-39; human NOX2 forward, 59-GTC TGG TAT TAC CGG GTT TA-39, and reverse, 59-GTG CTA CTG AAT AAG GAT CAG- 39; human NOX4 forward, 59-ATG GCT GTG TCC TGG AGG AG-39, reverse, 59-GAT CAT GAG GAA TAG CAC CA-39; human SOD1 forward, 59-AAG GAC TGA CTG AAG GCC TG-39, and reverse, 59-AAG CCA AAC GAC TTC CAG CG-39; human SOD2 forward, 59-CAG GCA GCT GGC TCC GGT TT-39, and reverse, 59-TGC AGT GGA TCC TGA TTT GG-39; human SOD3 forward, 59-ATG CTG GCG CTA CTG TGT TC-39, and reverse, 59-TTC CCG TTC TCC ACG CTG GC-39; human catalase forward, 59-ACC AGA TGC AGC ACT GGA AG-39, and reverse, 59-GGG GGT GTT ATT TCC AA CGA-39; human G3PDH forward, 59-ACC ACA GTC CAT GCC ATC AC-39and reverse 59- TCC ACC ACC CTG TTG CTG TA-39. Senescence associated b-galactosidase (SA b-gal) staining SA beta-gal was determined by using a senescence detection kit (Abcam) according to the manufacturer’s instructions. Briefly, cells were incubated overnight in freshly prepared staining solution (containing 1 mg/ml X-gal) at 37uC. The percentage of senescent cells was obtained by counting the number of blue-stained cells and the total cells per field under an inverted microscope. Gene silencing by stealth siRNA HUVECs or HAECs were transfected with synthetic siRNAs in Lipofectamine RNAi max containing Opti-MEM I at a final concentration of 10 nmol/L. At 12 hour post-transfection, the cell culture medium was replaced with growth medium; and the cells were then incubated for and additional 12 hours prior to use in experiments. Specific gene silencing was verified by RT-PCR and Western blot analysis. The nucleotide sequences of stealth siRNAs used in this study are as follow: for human VASH1 and its control, 59-CAA GGA CCG GAA GAA GGA UGU UUC U-39 and 59- CAA CCA AGG AGA GGA GUA UUG GUC U-39; for human HuR and its control, 59-CGG GAU AAA GUA GCA GGA CAC AGC U -39 and 59-CGG AAA UGA UGG GAC CAC AAA GGC U -39; for human SIRT1 and its control, 59-CAG GUU GCG GGA AUC CAA AGG AUA A-39 and 59-CAG GCG UAA GGA CCU GGA AAU GUA A-39; and for human SOD2 and its control, 59-GAG GAG AAC TCG CTT CGT ATT TGT A-3 and its control, 59-TAC TCA AAT ACG AAG CGA GTT CCU C-39. Immunocytochemical analysis HUVECs on culture slides were transfected with VASH1 siRNA or control siRNA. At the desire times thereafter the cells were fixed with 4% paraformaldehyde at room temperature, and then rendered permeable with 0.1% NP-40 in PBS. Nonspecific binding sites were blocked with 1% BSA in PBS. Primary antibody reactions were performed overnight at 4uC with anti-LC-3 antibody at a dilution of 1:100. Secondary antibody reactions were performed for 1 hour at room temperature with Alexa 488- conjugated goat IgG against mouse antibody (Molecular Probes, Eugene, OR) at a 1:100 dilution with 1 mmol/L To-Pro-3 iodine PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46459