Figure 3. 14-3-3s-enhanced CCE proliferation is suppressed by b-catenin siRNA. (A) CCE cells were transfected with scramble or the reported b-catenin siRNA sequence and co-transfected with 14-3-3s vectors for 48 h. b-catenin proteins were determined by Western blot analysis. (B) Viable cells were determined by trypan blue assay. Error bar is mean 6 s.d. (n = 3). *p,0.05; **p,0.01. doi:10.1371/journal.pone.0040193.g003 (Figure 1D) at 48 h. The inhibitory effect of 14-3-3s knockdown was confirmed by another siRNA sequence (Figure S1). We next evaluated the influence of 14-3-3 overexpression on CCE pro- liferation. CCE cells were transfected with each isoform of 14-3-3 and the isoform protein overexpression was verified by Western blotting (Figure 2A). Only 14-3-3s overexpression increased CCE cell numbers (Figure 2B). Furthermore, overexpression of 14-3-3s significantly increased viable cell numbers (Figure 2C, upper panel) and BrdU incorporation (Figure 2D) and partially rescued RA-induced decline in cell number (Figure 2C, lower panel). To ensure that the involvement of 14-3-3s in mESC proliferation is not limited to CCE, we evaluated the effect of 14-3-3s in other mESC proliferation. Overexpression of 14-3-3s in R1 and D3 mESC significantly increased viable cell number and BrdU incorporation to an extent comparable to CCE cells (Figure S2). Taken together, these results indicate that 14-3-3s plays a critical role in CCE proliferation. 14-3-3s Promotes mESC Proliferation via b-catenin To determine whether b-catenin is involved in 14-3-3s- mediated mESC proliferation, CCE cells were co-transfected with 14-3-3s overexpression vector and b-catenin siRNA. Suppression of b-catenin expression in CCE transfected with a previously described siRNA [30,31] (Figure 3A) resulted in abrogation of 14- 3-3s-induced cell number increases (Figure 3B). These results were confirmed by another siRNA sequence of b-catenin which exhibited a lesser effect on suppressing b-catenin and a correlated lesser effect on reducing CCE proliferation (Figure S3). To 14-3-3s Induces Mouse ES Cell Proliferation investigate the relationship between 14-3-3s and b-catenin, we transfected CCE cells with 14-3-3s and analyzed b-catenin phosphorylation, ubiquitination, degradation and nuclear trans- location. Phosphorylation and ubiquitination of b-catenin were significantly decreased (Figure 4A and 4B), whereas total b-catenin protein level was increased (Figure 4C, upper panel) in 14-3-3s overexpressed CCE cells. Furthermore, b-catenin protein levels were increased in the nuclear fraction (Figure 4C, lower panel). Thus, 14-3-3s prevents b-catenin from phosphorylation, ubiqui- tination and degradation, thereby increasing b-catenin protein stability and nuclear translocation. To ensure that nuclear b-catenin is active in promoting b- catenin/Tcf-targeted gene expression in mESCs, we analyzed b- catenin transcriptional activity by using TOPFLASH/FOP- FLASH reporters. 14-3-3s overexpression increased the pro- moter activity by ,5-fold over the control (Figure 4D). We next determined the expression of cyclin D1, one of the b-catenin target genes that are critical in cell cycle progression and cell proliferation. Cyclin D1 protein level was increased by ,3-fold in 14-3-3s-overexpressed cells (Figure 4E). Taken together, the results indicate that 14-3-3s promotes mESC proliferation via b- catenin. 14-3-3s Interacts with GSK-3b and Suppresses GSK-3b Activity We performed immuneprecipitation (IP) to determine interac- tion of 14-3-3s with GSK-3b as well as proteins in the b-catenin destruction complex. CCE cells were transfected with Flag-tagged 14-3-3s, 14-3-3s protein complex was pulled down by IP using a Flag antibody and interacting proteins were analyzed with Western blotting. 14-3-3s was co-immunoprecipitated with GSK- 3b but not b-catenin, axin or APC (Figure 5A. upper panel). 14-3- 3s was also co-immunoprecipitated with GSK-3b in R1 and D3 mESC (Figure 5A, middle and lower panels). To determine whether GSK-3b selectively interacts with 14-3-3s isoform, we transfected CCE with each Flag-tagged 14-3-3 isoform, immuno- precipitated the complex with Flag antibody and analyzed GSK- 3b protein by Western blotting. GSK-3b was co-precipitated only with 14-3-3s (Figure 5B). These results suggest that 14-3-3s binds GSK-3b in 14-3-3s transfected CCE cells. Binding and catalytic activities of GSK-3b are regulated by phosphorylation. We prepared GSK-3b mutants for potential 14-3-3 binding sites at Ser9 (S9A) or Thr309 (T309A), constructed the mutant into the expression vector and analyzed the influence of mutant over- expression on interaction with 14-3-3s. 14-3-3s interaction with GSK-3b was reduced by S9A mutation but not T309A (Figure 5C). These results suggest that 14-3-3s binding of GSK- 3b requires phosphorylation at Ser9. Since PI-3K/Akt pathway is involved in GSK-3b Ser9 phosphorylation, we evaluated the effect of wortmannin, a PI-3K inhibitor on interaction of GSK-3b with 14-3-3s. Wortmannin blocked the interaction in a concentration- dependent manner (Figure 5D). We suspected that 14-3-3s binding of GSK-3b might result in GSK-3b sequestration and inactivation. To address this, we analyzed the level of phosphorylated GSK-3b (pGSK-3b) and GSK-3b catalytic activity in 14-3-3s overexpressed CCE cells. Compared to non-transfected cells, GSK-3b catalytic activity was significantly reduced in 14-3-3s transfected cells (Figure 5E). In accordance with reduction of the catalytic activity, pGSK-3b was enhanced by 14-3-3s overexpression which was abrogated by wortmannin induced Akt dephosphorylation and inactivation (Figure 5F). These results indicate that 14-3-3s overexpression enhances GSK-3b phosphorylation and induces 14-3-3s binding PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e40193