14-3-3s Induces Mouse ES Cell Proliferation Figure 8. A schematic model illustrating the role of 14-3-3s in regulating ES cell proliferation via GSK-3b/b-catenin pathway. doi:10.1371/journal.pone.0040193.g008 for 14-3-3s binding of GSK-3b. GSK-3b phosphorylation at Ser9 and Thr309 are potential residues for 14-3-3 interaction. Mutation of GSK-3b Ser9 abrogates GSK-3b binding to 14-3- 3s whereas mutation of Thr309 did not alter its binding. These results suggest that PI-3K/Akt plays an essential role in promoting 14-3-3s binding and sequestration of GSK-3b by Ser9 phosphorylation. PI-3K/Akt is recognized as an important signaling pathway for promoting ESC proliferation. We demon- strate that enhanced 14-3-3s/GSK-3b interaction is an impor- tant downstream mechanism by which PI-3K/Akt mediates ESC proliferation and renewal. It is interesting to note that b-catenin siRNA does not completely abrogate the enhancing action of 14-3-3s on cell proliferation (Figure 3B). It is possible that 14-3-3s may enhance cell proliferation by multiple mechanisms. Besides the Wnt/b- catenin mechanism, 14-3-3s may bind phosphorylated Raf-1, activate Raf-1 and its downstream signaling pathway [41,42]. Moreover, 14-3-3 was found to regulate the mammalian target of rapamycin (mTOR) pathway by interacting with tuberous sclerosis complex 2 (TSC2), and sequestering it from binding to mTOR complex, thereby increasing the mTOR activity on de novo protein synthesis and cell proliferation [43,44]. In summary, this study shows for the first time that 14-3-3s regulates mESC proliferation by binding and sequestering GSK- 3b as well as inducing GSK-3b phosphorylation and inactivation in a PI-3K/Akt-dependent manner. 14-3-3s is a novel target for ES cell expansion. Supporting Information Figure S1 14-3-3s knockdown suppressed cell prolifer- ation. CCE cells were transfected with scramble or 14-3-3s siRNA (Invitrogen, sence: GCGCAUCAUCGAU UCUGCCCG- GUCA; antisence: UGACCGGGCAGAAUCGAUGAUGCGC) for 24 h or 48 h. (A) Knockdown of 14-3-3s expression was determined by Western blot analysis after transfection for 48 h. (B) Proliferation and viable cell numbers of CCE cells were determined by trypan blue assay, and (C) BrdU assay. Each bar represents mean 6 s.d. (n = 3). N.S. denotes statistically not significant; *, p,0.05. (EPS) Figure S2 14-3-3s transfection increased (A) cell num- bers (B) BrdU incorporation in R1 and D3 mES cells. Each bar is mean 6 s.d. of three independent experiments. * p,0.05. (EPS) Figure S3 b-catenin knockdown suppressed 14-3-3s- enhanced cell proliferation. CCE cells were co-transfected with control or 14-3-3s overexpression vectors, and scramble or b-catenin siRNA (Invitrogen, sence: CCCAGAAUGCC- GUUCGCCUUCAUUA; antiscene: UAAUGAAGGC- GAACGGCAUUCUGGG) for 48 h. (A) Protein level of reduced b-catenin was confirmed by Western blot analysis after trans- fection for 48 h. (B) Proliferation of CCE cells was determined by trypan blue assay after 48 h transfection. Each bar represents mean 6 s.d. (n = 3). *, p,0.05; **, p,0.01. (EPS) PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e40193