14-3-3s Induces Mouse ES Cell Proliferation Figure 2. 14-3-3s overexpression increases CCE proliferation. (A) and (B) CCE cells were transfected with control (CTR) or each 14-3-3 isoform and (A) all isoform proteins were analyzed by Western blotting, (B) cell numbers were counted. (C) CCE cells transfected with CTR or 14-3-3s vectors were treated with (lower panel) or without RA (upper panel) and cell number were counted. (D) CCE cells transfected with CTR or 14-3-3s vectors and BrdU incorporation were determined. Error bars are mean 6 s.d. (n = 3). N.S. denotes statistically not significant; *p,0.05; **p,0.01. doi:10.1371/journal.pone.0040193.g002 for 46 h, then incubated with MG-132 (10 mM) for an additional 2 h. Cell lysates were harvested, immunoprecipitated with a mouse monoclonal anti-HA antibody, and immunoblotted with rabbit polyclonal antibodies against ubiquitin (Cell Signaling). GSK-3b Activity Assay Assay of GSK-3b activity was based on measuring tau phosphorylation at Ser-396 and Ser-199 [36,37] by using an ELISA kit (Invitrogen). In brief, CCE cells were co-transfected for 48 h with GSK-3b expression vector and 14-3-3s-Flag vectors or their respective control vectors. The transfected CCE cells were lysed with RIPA buffer and immunoprecipitated with a specific antibody against GSK-3b. The immunoprecipitate was washed and incubated with an assay buffer containing 100 mM ATP and recombinant Tau proteins, then with an antibody against phospho-Ser396 of Tau, a secondary antibody, and substrates. The reaction was terminated by adding a stop reagent, and the optical density of the sample was analyzed at 450 nm in an ELISA reader. Values of Tau phosphor-Ser396 were normalized to the total protein level of Tau. Statistical Analysis Differences between groups were analyzed by Student t test. A p value less than 0.05 was considered statistically significant. Results 14-3-3s Promotes Mouse ES Cell (mESC) Proliferation Retinoic acid (RA) is known to induce differentiation and suppress proliferation of mESC [38,39]. However, the underlying mechanism is not entirely clear. To investigate whether 14-3-3 proteins are involved in regulating the actions of RA, we analyzed 14-3-3 isoforms in RA-treated mESCs. Among seven 14-3-3 isoforms, only 14-3-3s was significantly reduced by RA (Figure 1A). To determine the role of 14-3-3s in mESC proliferation, we treated CCE cells with two 14-3-3s siRNA sequences. Using a previously reported sequence [28,29], our results confirm that 14-3-3s siRNA specifically suppressed 14-3- 3s proteins without changing the expression of other 14-3-3 isoforms (Figure 1B). Knockdown of 14-3-3s resulted in reduced CCE viable cell numbers (Figure 1C) and BrdU incorporation PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e40193