ES cell renewal and differentiation. Its activity must be tightly regulated. However, little is known about its regulation in ES cells. We propose in this study that 14-3-3 proteins regulate GSK-3b availability. 14-3-3 proteins are 28- to 33-kDa acidic polypeptides found in all eukaryotic organisms [16–18]. 7 members (b, c, e, g, s, h/t and f) are found in mammals. These isoforms form homo- or hetero-dimers to serve as scaffolds. At least 200 proteins are reported to interact with 14-3-3 [16]. Through binding to various classes of proteins including enzymes, transcription factors, cytoskeletal proteins, signaling molecules, apoptosis factors and tumor suppressors, 14-3-3 proteins are involved in diverse cellular functions and pathophysiological processes [17]. 14-3-3 isoforms have been reported to regulate GSK-3b. 14-3-3f was reported to bind GSK-3b, and stimulates tau phosphorylation in the brain [19]. 14-3-3b interacts with Ser9-phosphorylated GSK-3b to control neuronal survival [20]. 14-3-3 was also reported to interact with b-catenin and modify its transcriptional activity. 14-3-3f interacts with b-catenin and enhances b-catenin transactivation action [21]. On the other hand, 14-3-3f was reported to interact with Chibby protein to export b-catenin from nucleus and consequently attenuate the b-catenin transcriptional activity [22]. These results indicate that 14-3-3 proteins are functionally complex. Little is known about 14-3-3 proteins in ES cells, let alone their roles in ES cell renewal and proliferation. In this study, we investigated the involvement of 14-3-3 proteins in regulating mouse ES cell (mESC) proliferation. We provide evidence that 14- 3-3s isoform regulates mESC proliferation by binding and sequestering GSK-3b and enhancing Wnt3a-induced GSK-3b phosphorylation and inactivation. 14-3-3s overexpression rescues retinoid acid (RA)-induced growth arrest by increasing GSK-3b phosphorylation and b-catenin level. Materials and Methods Cell Culture and Reagents CCE, a mESC line derived from the 129/Sv mouse strain, was obtained from StemCell Technologies with permission from Drs. Robertson and Keller (Vancouver, Canada). CCE cells were cultured on gelatin-coated dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 10 ng/mL leukemia inhibitory factor at 37uC in a humidified 5% CO2 atmosphere [23,24]. D3 and R1 mouse ES cells [25,26] were cultured and maintained on feeder cells comprising mitotically inactivated primary mouse embryonic fibroblasts (MEFs) in the same medium of CCE cells. Mouse recombinant Wnt3a and Wnt inhibitor, DKK-1 were from Calbiochem (San Diego, CA, USA). The PI3-K inhibitor wortmannin was from Sigma (St. Louis, MO, USA). Plasmid Constructs and Transfection cDNA of each 14-3-3 isoform (b, c, e, g, s, h and f) was amplified by PCR and cloned into the p3XFlag-CMV expression vector (Sigma) with the restriction enzymes HindIII and BamH1. The expression vector of b-catenin was constructed as described [27]. Small interfering RNA (siRNA) of 14-3-3s and b-catenin was from Santa Cruz Biotechnology (Santa Cruz, CA). 14-3-3s siRNA used for knockdown experiments comprised three RNA sequences including sc-29591A (sense: GAAGACAUGGCAG- CUUUCATT, antisense: UGAAAGCUGCCAUGUCUUCTT); sc-29591B (sense: CCCAAACCCUGAAUGUUCATT, anti- sense: UGAACAUUCAGGGUUUGGGTT) and sc-29591C 14-3-3s Induces Mouse ES Cell Proliferation (sense: GUGUGACCAUGGUACCAAUTT, antisense: AUUG- GUAC CAUGGUCACACTT) [28,29]. The siRNA specific to b- catenin was sc-29209 (sense: AGCUGAUAUUGAUGGA- CAGTT and antisense: CUGUCCAUCAAUAUCAGCUTT) [30,31]. Expression vectors containing GSK-3b wild-type (WT) and GSK-3b Ser9A mutant cDNA were from Addgene (plasmid 16260 and 16261) [32]. The GSK-3b Thr309A mutant cDNA plasmid was cloned using a site-directed mutagenesis kit (Stratagene, La Jolla, USA). Expression vectors and siRNA were co-transfected into mESCs using Effectene transfection reagent (Qiagen, GmbH, Hilden, Germany) as described [33,34]. In brief, DNA plasmids or siRNA were mixed with Enhancer and Effectene at a ratio of 1 (mg) to 1.6 (mL) to 4 (mL). The adherent CCE colonies were trypsinized to yield single-cell suppression. The D3 or R1 cells on MEFs were trypsinized and pre-precipitated for 1 h to remove feeder cells. DNA (or siRNA)-Effectene mixture was added to CCE, D3 or R1 cell suspension, which was seeded on gelatin-coating plates or MEF feeder cells for 24–48 h. Cells were harvested and assayed. Western Blot Analysis Western blot analysis was performed as described [35]. Briefly, cells were washed with phosphate-buffered saline (PBS) and lysed in ice-cold RIPA lysis buffer (Upstate, Lake Placid, NY) containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Man- nheim, Germany). The lysate was centrifuged, the supernatant collected, and protein concentration determined by the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). 30 mg of supernatant proteins was applied to each lane of an SDS polyacrylamide gel. Proteins were resolved by electrophoresis and transferred to PVDF membrane (Millipore, Bedford, MA). Membranes were blocked with 5% milk, incubated with primary antibodies overnight at 4uC, washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The protein bands were visualized by enhanced chemiluminescence (PerkinElmer, Shelton, CT). Rabbit polyclonal antibodies against b-catenin, phosphor-b-catenin, GSK-3b, phospho-GSK-3b (Ser9), Akt, phospho-Akt, APC, axin, ubiquitin and cyclin D1 were from Cell Signaling. Mouse monoclonal or rabbit polyclonal antibodies against 14-3-3 iso- forms, Oct3/4 and lamin A/C were from Santa Cruz Bio- technology. Antibodies specifically for 14-3-3 isoforms were raised against peptide regions or sequences as follows: C-terimal region of 14-3-3b, amino acids 130–170 of 14-3-3e, 206–246 of 14-3-3c, 205–245 of 14-3-3h, 172–202 of 14-3-3g, 109–149 of 14-3-3f and the entire recombinant protein of 14-3-3s. Mouse monoclonal antibodies against Flag, HA and actin were from Sigma. Preparation of Nuclear Proteins Nuclear proteins were extracted by using an extraction kit (Chemicon). Briefly, cells were harvested and lysed with cytoplas- mic buffer containing protease inhibitors for 15 min at 4uC, mixed, and centrifuged at 8,000 g for 20 min at 4uC. Supernatants were collected (cytoplasmic extraction) and pellets were resus- pended in nucleus buffer containing protease inhibitors for 15 min at 4uC. The resuspended sample was mixed and centrifuged at 16,000 g for 10 min at 4uC. The supernatant containing nuclear extraction proteins was collected and stored at 280uC. Cell Proliferation Analysis For cell proliferation analysis in this study, 1.56105 transfected cells were seeded (defined as 0 h) and incubated for the indicated time. Cell number was determined by trypan blue assay. Cells were trypsinized, resuspended in medium, and viable cells were PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e40193