(Molecular Devices, Tokyo, Japan) with excitation at 480 nm and emission at 530 nm. Mouse model of acute lung injury Paraquat treatment. Wild-type (WT) mice (Charles River Japan, Yokohama) and C57 BJ/6 background VASH1 (+/2) KO mice [3,10] of C57 BJ/6 background (9 to 10 weeks of age) were used in this study. Paraquat was dissolved in phosphate-buffered saline (5 mg/mL) and injected intraperitoneally at the dosage of 50 mg/kg body weight [11]. To evaluate the protective effect of VASH1, we used purified AdVASH1. Purified AdLacZ was used for the control [2,12]. Mice were anesthetized with ketamine (0.1 mg/g) and xylazine (0.01 mg/g) administered by intraperito- neal injection and then intubated orotracheally with a 22-G angiocatheter. A total of 75 microl of AdVASH1 or AdLacZ, containing 16109 plaque-forming units, was injected into each mouse ototracheally [13]. Paraquat was injected intraperitoneally at the dosage of 50 mg/kg body weight 72 hours after adminis- tration of the adenovirus. The mice were followed periodically over 10 days, and the number of deaths was recorded. At the end of 10 days, all remaining mice were terminated. b-gal staining. Mice were sacrificed 2 days after the intratracheal administration of PBS or AdLacZ. The pulmonary circulation was flushed with ice-cold 2% buffered paraformalde- VASH1 Enhances Stress Resistance of Endothelium hyde, 0.2% glutaraldehyde, and 0.02% NP40 via the right ventricle. The lung blocks were inflation-fixed through the trachea with 2% buffered paraformaldehyde/PBS for 2 hours at room temperature. Thereafter, the lungs were incubated at room temperature in 1 mg/mL X-gal, 5 mmol/L potassium ferricya- nide crystalline, 5 mmol/L potassium ferricyanide trihydrate, 2 mmol/L magnesium chloride, and 0.1% NP40 and then embedded in paraffin. Four-micrometer sections were prepared and then counterstained with nuclear fast red. Histological analysis of the lungs. Mice were sacrificed 2 days after paraquat administration. The lungs were inflation-fixed through the trachea with 4% buffered paraformaldehyde/PBS overnight at 4uC, and then embedded in paraffin. Four- micrometer sections were prepared and then stained with hematoxylin and eosin (H&E). For the staining of 8-OHdG, sections were incubated with the anti-8-OHdG antibody overnight at 4uC at a 1:20 dilution. They were then incubated in 10% H2O2/methanol to block endogenous peroxidase activity. The secondary antibody reaction was performed with biotin-conjugated anti-mouse IgG for 40 min at room temperature. Streptavidin-biotin peroxidase complex for- mation was performed for 30 min at room temperature. The peroxidase products were visualized by using diaminobenzidine. Figure 2. Overexpression of VASH1 inhibits premature senescence and cell death of HUVECs induced by cellular stresses. (A) HUVECs were infected with AdVASH1 or AdLacZ. After a 24-hour incubation, RT-PCR and Western blotting for VASH1 were performed. (B) HUVECs infected with AdVASH1 or AdLacZ were exposed to 100 mmol/L H2O2 for 1 hour, followed culture for 24 hours (on the left) or to 0% FCS/aMEM 24 hours (on the right). After a 6-day culture, SA b-gal staining was performed. Scale bars are 250 microm. SA b-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P,0.01, N = 3). (C) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, HUVECs were exposed to 100 mmol/L H2O2 or to 0% FCS/aMEM for 24 hours, and then the trypan blue exclusion assay was performed. Blue-stained cells were quantified, and the % of dead cells was calculated (*P,0.01, N = 3). (D) HUVECs infected with AdVASH1 or AdLacZ were exposed to 100 mmol/L H2O2 for 48 hours or to 0% FCS/aMEM for 24 hours for 24 hours; and then the trypan blue exclusion assay was performed (*P,0.01, N = 3). (E) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, the growth medium was replaced with 50% conditioned medium derived from mock or VASH1 over-expressing MS1 clone 4. After a 24-hour incubation, the trypan blue exclusion assay was performed (*P,0.01, N = 3). All the studies were repeated at least 3 times to confirm the reproducibility. doi:10.1371/journal.pone.0046459.g002 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46459