Acknowledgments We thank Ms. Ya-Lin Hung and Nathalie Huang for helpful assistance. References 1. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156. 2. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78: 7634–7638. 3. Bouhon IA, Joannides A, Kato H, Chandran S, Allen ND (2006) Embryonic stem cell-derived neural progenitors display temporal restriction to neural patterning. Stem Cells 24: 1908–1913. 4. Sakurai H, Okawa Y, Inami Y, Nishio N, Isobe K (2008) Paraxial mesodermal progenitors derived from mouse embryonic stem cells contribute to muscle regeneration via differentiation into muscle satellite cells. Stem Cells 26: 1865– 1873. 5. Liu N, Lu M, Tian X, Han Z (2007) Molecular mechanisms involved in self- renewal and pluripotency of embryonic stem cells. J Cell Physiol 211: 279–286. 6. Okita K, Yamanaka S (2006) Intracellular signaling pathways regulating pluripotency of embryonic stem cells. Curr Stem Cell Res Ther 1: 103–111. 7. Katoh M (2007) WNT signaling pathway and stem cell signaling network. Clin Cancer Res 13: 4042–4045. 8. Kleber M, Sommer L (2004) Wnt signaling and the regulation of stem cell function. Curr Opin Cell Biol 16: 681–687. 9. Doble BW, Woodgett JR (2003) GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116: 1175–1186. 10. Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303: 1483–1487. 11. Zeng X, Tamai K, Doble B, Li S, Huang H, et al. (2005) A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438: 873–877. 12. Aubert J, Dunstan H, Chambers I, Smith A (2002) Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 20: 1240–1245. 13. Kielman MF, Rindapaa M, Gaspar C, van Poppel N, Breukel C, et al. (2002) Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat Genet 32: 594–605. 14. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10: 55–63. 15. Welsh GI, Wilson C, Proud CG (1996) GSK3: a SHAGGY frog story. Trends Cell Biol 6: 274–279. 16. Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40: 617–647. 17. Morrison DK (2009) The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends Cell Biol 19: 16–23. 18. Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, et al. (1997) The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91: 961–971. 19. Agarwal-Mawal A, Qureshi HY, Cafferty PW, Yuan Z, Han D, et al. (2003) 14- 3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule- associated tau phosphorylation complex. J Biol Chem 278: 12722–12728. 20. Mwangi S, Anitha M, Fu H, Sitaraman SV, Srinivasan S (2006) Glial cell line- derived neurotrophic factor-mediated enteric neuronal survival involves glycogen synthase kinase-3beta phosphorylation and coupling with 14-3-3. Neuroscience 143: 241–251. 21. Tian Q, Feetham MC, Tao WA, He XC, Li L, et al. (2004) Proteomic analysis identifies that 14-3-3zeta interacts with beta-catenin and facilitates its activation by Akt. Proc Natl Acad Sci U S A 101: 15370–15375. 22. Li FQ, Mofunanya A, Harris K, Takemaru K (2008) Chibby cooperates with 14-3-3 to regulate beta-catenin subcellular distribution and signaling activity. J Cell Biol 181: 1141–1154. 14-3-3s Induces Mouse ES Cell Proliferation Author Contributions Conceived and designed the experiments: JYL KKW TCC. Performed the experiments: TCC CCL EWH SML. Analyzed the data: JYL KKW TCC BSK. Contributed reagents/materials/analysis tools: YHC SPL LYS TLS BLY SFY. Wrote the paper: JYL KKW. 23. Chang TC, Chen YC, Yang MH, Chen CH, Hsing EW, et al. (2010) Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner. PLoS One 5: e9187. 24. Liou JY, Ellent DP, Lee S, Goldsby J, Ko BS, et al. (2007) Cyclooxygenase-2- derived prostaglandin e2 protects mouse embryonic stem cells from apoptosis. Stem Cells 25: 1096–1103. 25. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87: 27–45. 26. Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A (1993) Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87–89. 27. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, et al. (1997) Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC2/ 2 colon carcinoma. Science 275: 1784–1787. 28. Wilker EW, Grant RA, Artim SC, Yaffe MB (2005) A structural basis for 14-3- 3sigma functional specificity. J Biol Chem 280: 18891–18898. 29. Yang HY, Wen YY, Chen CH, Lozano G, Lee MH (2003) 14-3-3 sigma positively regulates p53 and suppresses tumor growth. Mol Cell Biol 23: 7096– 7107. 30. Liu P, Yang J, Pei J, Pei D, Wilson MJ (2010) Regulation of MT1-MMP activity by beta-catenin in MDCK non-cancer and HT1080 cancer cells. J Cell Physiol 225: 810–821. 31. Yan W, Tai HH (2006) Glycogen synthase kinase-3 phosphorylation, T-cell factor signaling activation, and cell morphology change following stimulation of thromboxane receptor alpha. J Pharmacol Exp Ther 317: 267–274. 32. Zhou BP, Deng J, Xia W, Xu J, Li YM, et al. (2004) Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 6: 931–940. 33. Ko BS, Chang TC, Shyue SK, Chen YC, Liou JY (2009) An efficient transfection method for mouse embryonic stem cells. Gene Ther 16: 154–158. 34. Liou JY, Ko BS, Chang TC (2010) An efficient transfection method for mouse embryonic stem cells. Methods Mol Biol 650: 145–153. 35. Liou JY, Ghelani D, Yeh S, Wu KK (2007) Nonsteroidal anti-inflammatory drugs induce colorectal cancer cell apoptosis by suppressing 14-3-3epsilon. Cancer Res 67: 3185–3191. 36. Ding Q, He X, Xia W, Hsu JM, Chen CT, et al. (2007) Myeloid cell leukemia-1 inversely correlates with glycogen synthase kinase-3beta activity and associates with poor prognosis in human breast cancer. Cancer Res 67: 4564–4571. 37. Ishiguro K, Shiratsuchi A, Sato S, Omori A, Arioka M, et al. (1993) Glycogen synthase kinase 3 beta is identical to tau protein kinase I generating several epitopes of paired helical filaments. FEBS Lett 325: 167–172. 38. Duester G (2008) Retinoic acid synthesis and signaling during early organogenesis. Cell 134: 921–931. 39. Martin-Ibanez R, Urban N, Sergent-Tanguy S, Pineda JR, Garrido-Clua N, et al. (2007) Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells. J Neurosci Res 85: 2686–2701. 40. Su YW, Hao Z, Hirao A, Yamamoto K, Lin WJ, et al. (2011) 14-3-3sigma regulates B-cell homeostasis through stabilization of FOXO1. Proc Natl Acad Sci U S A 108: 1555–1560. 41. Fantl WJ, Muslin AJ, Kikuchi A, Martin JA, MacNicol AM, et al. (1994) Activation of Raf-1 by 14-3-3 proteins. Nature 371: 612–614. 42. Li S, Janosch P, Tanji M, Rosenfeld GC, Waymire JC, et al. (1995) Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins. EMBO J 14: 685–696. 43. Li Y, Inoki K, Yeung R, Guan KL (2002) Regulation of TSC2 by 14-3-3 binding. J Biol Chem 277: 44593–44596. 44. Shumway SD, Li Y, Xiong Y (2003) 14-3-3beta binds to and negatively regulates the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product, tuberin. J Biol Chem 278: 2089–2092. PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e40193