(B) HUVECs were infected with AdVASH1 or AdLacZ. After a 24-hour incubation, SU5416 was added. After an additional 3 days’ incubation, the trypan blue exclusion assay was performed. Blue-stained cells were quantified, and the % of dead cells was calculated. *Significant difference compared with the value for the corresponding AdLacZ. (C) HUVECs were transfected with VASH1 siRNA or control siRNA. In some case, VEGF (1 nmol/L) was added to HUVECs that had been transfected with VASH1 siRNA. The cells were cultured for 3 days, and observed by phase-contrast microscopy. (TIFF) Figure S3 Intratracheal administration of adenovirus vector. (A) PBS (left photo) or AdVASH1 (right photo) was intratracheally administered to WT mice. Twenty-four hours after the administration, the lungs were removed and processed for b- gal staining. (B) Microscopic observation showed that SA b-gal- positive cells were found mainly in the bronchial epithelium, but also in some blood vessels and interstitial macrophages. (C) After the intratracheal administration of AdVASH1 to WT mice, the lungs were removed at the indicated time points; and tissue extracts prepared from them were then Western blotted for VASH1. (TIFF) References 1. Phng LK, Gerhardt H (2009) Angiogenesis: a team effort coordinated by notch. Dev Cell 16: 196–208. 2. Watanabe K, Hasegawa Y, Yamashita H, Shimizu K, Ding Y, et al. (2004) Vasohibin as an endothelium-derived negative feedback regulator of angiogen- esis. J Clin Invest 114: 898–907. 3. Kimura H, Miyashita H, Suzuki Y, Kobayashi M, Watanabe K, et al. (2009) Distinctive localization and opposed roles of vasohibin-1 and vasohibin-2 in the regulation of angiogenesis. Blood 113: 4810–4818. 4. Andreassi MG (2009) Metabolic syndrome, diabetes and atherosclerosis: influence of gene-environment interaction. Mutat Res 667: 35–43. 5. Erusalimsky JD, Kurz DJ (2005) Cellular senescence in vivo: its relevance in ageing and cardiovascular disease. Exp Gerontol 40: 634–642. 6. Minamino T, Komuro I (2008) Role of telomeres in vascular senescence. Front Biosci 13: 2971–2979. 7. O’Riordan E, Mendelev N, Patschan S, Patschan D, Eskander J, et al. (2007) Chronic NOS inhibition actuates endothelial-mesenchymal transformation. Am J Physiol Heart Circ Physiol 292: H285–294. 8. Miyashita H, Suzuki H, Ohkuchi A, Sato Y (2011) Mutual Balance between Vasohibin-1 and Soluble VEGFR-1 in Endothelial Cells. Pharmaceuticals 4: 782–793. 9. Namba K, Abe M, Saito S, Satake M, Ohmoto T, et al. (2000) Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31. Oncogene 19: 106–114. 10. Hosaka T, Kimura H, Heishi T, Suzuki Y, Miyashita H, et al. (2009) Vasohibin- 1 expression in endothelium of tumor blood vessels regulates angiogenesis. Am J Pathol 175: 430–439. 11. Jang YC, Perez VI, Song W, Lustgarten MS, Salmon AB, et al. (2009) Overexpression of Mn superoxide dismutase does not increase life span in mice. J Gerontol A Biol Sci Med Sci 64: 1114–1125. 12. Yamashita H, Abe M, Watanabe K, Shimizu K, Moriya T, et al. (2006) Vasohibin prevents arterial neointimal formation through angiogenesis inhibi- tion. Biochem Biophys Res Commun 345: 919–925. 13. DuPage M, Dooley AL, Jacks T (2009) Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4: 1064– 1072. 14. Zhan H, Suzuki T, Aizawa K, Miyagawa K, Nagai R (2010) Ataxia telangiectasia mutated (ATM)-mediated DNA damage response in oxidative stress-induced vascular endothelial cell senescence. J Biol Chem 285: 29662– 29670. 15. Rajawat YS, Hilioti Z, Bossis I (2009) Aging: central role for autophagy and the lysosomal degradative system. Ageing Res Rev 8: 199–213. 16. Suzuki Y, Kobayashi M, Miyashita H, Ohta H, Sonoda H, et al. (2010) Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion. J Cell Sci 123: 3094–3101. 17. Hinman MN, Lou H (2008) Diverse molecular functions of Hu proteins. Cell Mol Life Sci 65: 3168–3181. 18. Abdelmohsen K, Lal A, Kim HH, Gorospe M (2007) Posttranscriptional orchestration of an anti-apoptotic program by HuR. Cell Cycle 6: 1288–1292. 19. Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, et al. (2007) Autocrine VEGF signaling is required for vascular homeostasis. Cell 130: 691–703. VASH1 Enhances Stress Resistance of Endothelium Figure S4 VASH1 does not increase stress resistance of NHBECs. (A) Expression of endogenous VASH1 in HUVECs and NHBECs was analyzed by RT-PCR. (B) NHBECs were infected with AdVASH1 or AdLacZ. After a 72-hour incubation, Western blotting for VASH1, SOD2, and SIRT1 was performed. (C) NHBECs were infected with AdVASH1 or AdLacZ. After a 72-hour incubation, NHBECs were exposed to 400 mmol/L H2O2 for 24 hours; and the trypan blue exclusion assay was then performed. Blue-stained cells were quantified, and the % of dead cells was calculated. Values are the ratio of blue-stained cells to total cells, and are the means and SDs of 4 wells. (TIFF) Ackowledgments We thank Ms. Yuriko Fujinoya for her excellent technical assistance. Author Contributions Conceived and designed the experiments: Y. Sato. Performed the experiments: HM TW HH. Analyzed the data: YH YO TK. Contributed reagents/materials/analysis tools: Y. Suzuki TN SI MO. Wrote the paper: HM TW Y. Sato. 20. Bertram C, Hass R (2008) Cellular responses to reactive oxygen species-induced DNA damage and aging. Biol Chem 389: 211–220. 21. Potente M, Dimmeler S (2008) Emerging roles of SIRT1 in vascular endothelial homeostasis. Cell Cycle 7: 2117–22. 22. Gorospe M, de Cabo R (2008) AsSIRTing the DNA damage response. Trends Cell Biol 18: 77–83. 23. Tomita M, Okuyama T, Katsuyama H, Miura Y, Nishimura Y, et al. (2007) Mouse model of paraquat-poisoned lungs and its gene expression profile. Toxicology 231: 200–209. 24. Gurevich F, Perazella MA (2009) Renal effects of anti-angiogenesis therapy: update for the internist. Am J Med 122: 322–328. 25. Heishi T, Hosaka T, Suzuki Y, Miyashita H, Oike Y, et al. (2010) Endogenous angiogenesis inhibitor vasohibin1 exhibits broad-spectrum antilymphangiogenic activity and suppresses lymph node metastasis. Am J Pathol 176: 1950–1958. 26. Saito D, Maeshima Y, Nasu T, Yamasaki H, Tanabe K, et al. (2011) Amelioration of renal alterations in obese type 2 diabetic mice by vasohibin-1, a negative feedback regulator of angiogenesis. Am J Physiol Renal Physiol 300: F873–886. 27. Shimizu K, Watanabe K, Yamashita H, Abe M, Yoshimatsu H, et al. (2005) Gene regulation of a novel angiogenesis inhibitor, vasohibin, in endothelial cells. Biochem Biophys Res Commun 327: 700–706. 28. Ushio-Fukai M, Alexander RW (2004) Reactive oxygen species as mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol Cell Biochem 264: 85–97. 29. Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal. 15:1583–1606. 30. Suzuki K, Tatsumi H, Satoh S, Senda T, Nakata T, et al. (1993) Manganese- superoxide dismutase in endothelial cells: localization and mechanism of induction. Am J Physiol 265: H1173–1178. 31. Didion SP, Faraci FM (2005) Ceramide-induced impairment of endothelial function is prevented by CuZn superoxide dismutase overexpression. Arterio- scler Thromb Vasc Biol 25: 90–95. 32. Finkel T, Deng CX, Mostoslavsky R (2009) Recent progress in the biology and physiology of sirtuins. Nature 460: 587–591. 33. Orimo M, Minamino T, Miyauchi H, Tateno K, Okada S, et al. (2009) Protective role of SIRT1 in diabetic vascular dysfunction. Arterioscler Thromb Vasc Biol 29: 889–894. 34. Stein S, Lohmann C, Schafer N, Hofmann J, Rohrer L, et al. (2010) SIRT1 decreases Lox-1-mediated foam cell formation in atherogenesis. Eur Heart J 31: 2301–2309. 35. Takemura A, Iijima K, Ota H, Son BK, Ito Y, et al. (2011) Sirtuin 1 retards hyperphosphatemia-induced calcification of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 31: 2054–2062. 36. Zhang QJ, Wang Z, Chen HZ, Zhou S, Zheng W, et al. (2008) Endothelium- specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice. Cardiovasc Res 80: 191–199. 37. Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L, et al. (2007) SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev 21: 2644–2658. PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e46459