Sik1 Controls Cardiomyogenesis were identified (Fig. 1B, lanes 7, 8 and 9). The presence of the fusion protein in sik1flp/flp ES cells was confirmed by western blot analysis using an antibody against the b-galactosidase (Fig. 1C). Moreover, enzyme activity, determined as the ability of truncated sik1 to phosphorylate the specific AMARA peptide substrate in comparison to the full-length protein, was measured. To this end, FLAG-tagged sik1 (1–249) and full-length protein were expressed in HEK293 cells and, after immunoprecipitation, the peptide phosphorylation activity was measured using radio-labeled ATP. As expected, the full-length sik1 was able to efficiently phosphorylate the AMARA substrate; whereas the phosphorylation signal of the mutant protein was dramatically reduced, indicating that the catalytic activity of the truncated sik1 is almost inactive (Fig. 1D). We then evaluated the subcellular localization of the truncated form of sik1 (residues 1 to 249) produced by the trapped allele. It is known that the wild-type protein is resident in the nucleus where it exerts its function [16]. In contrast, the cells expressing the truncated form of sik1 showed an intense signal only in the cytoplasmic compartment, as opposed to the full-length protein that was mainly located in the nucleus (Fig. 1E). The gene-trap insertion resulted in the deletion of the nuclear localization signal; therefore, as expected, this truncated version of the sik1 protein showed a different localization compared to the full-length sik1. Altogether, these data indicate that the gene-trap insertion interrupts sik1 expression and results in the production of an inactive protein with a cytoplasmic localization. Delayed Cardiomyocyte Differentiation in sik1flp/flp ES Cells The next step was to evaluate the developmental potential of sik1flp/flp ES cells to differentiate in cardiomyocytes compared to wild-type and sik1+/flp cells using the ‘‘hanging drop’’ method. The embryoid bodies (EBs) generated clusters of adherent cells that began to contract rhythmically, indicating differentiation to the cardiac lineage, a derivative of the mesoderm layer. Seventy-six percent of plated EBs derived from wild-type ES cells showed areas of contracting cardiomyocytes starting from the day after adhesion (day 6, Fig. 2A). The appearance of beating EBs increased and reached over 92% at day 8 of culture. In contrast, EBs derived from sik1flp/flp cells showed a dramatic reduction in the formation of contractile areas (5% at day 6). However, a gradual increase of beating EBs was seen in the following 4 days of culture, Figure 2. In vitro differentiation and proliferation potential of sik1flp/flp ES cells. (A) Percentages of EBs differentiating to beating cardiomyocytes. EBs derived from wild-type, sik1wt/flp, sik1flp/flp and sik1flp/flp+HA-sik1 ES cells were used to monitor differentiation into cardiomyocytes. Sik1flp/flp + HAsik1 used corresponds to HAsik1 B6 clone expressing the highest level of sik1 (B). Three independent experiments were performed, each plating an equal number of EBs (n = 120). The statistical significance of the observed differences between the percentage of wild-type and mutant beating EBs were analyzed and the time points in which major differences were observed (days 6 and 7) showed significant p- values of 1.109e208 and 3.271e208, respectively. P-values were calculated using a two-sample test for equality of proportions with continuity correction. (B) RT- and Q-PCR analysis on wild-type ES cells and three representative clones carrying the HA-sik1 transgene. Data represent fold change of transgene expression vs. sik1 endogenous level. (C) Proliferation assay measured by BrdU incorporation. (D). Cell-cycle distribution of the wild-type and sik1flp/flp populations by FACS analysis. doi:10.1371/journal.pone.0009029.g002 PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9029