Sik1 Controls Cardiomyogenesis Figure 4. Measurement of myosin positive cells in wild-type and sik1flp/flp EBs by FACS. (A) EBs were analyzed by FACS at days 0, 5, 6, 7, 9, 11 and 12 of differentiation with the antibody MF20. Data represent results of one out of three independent experiments. (B) Graphic representation of FACS analysis. Data represent mean 6 S.E. of three biologically independent experiments (*, p,0.05 compared with wild-type EBs. P-values were calculated using a two-tailed, unpaired t test). (C) Graphic representation of low fluorescence vs high fluorescence MF20 positive cells distribution, as by FACS analysis (n = 3). Wild-type and sik1flpflp cells were used throughout the analysis. (D) Costaining experiment using anti-troponin and anti-BrdU to detect proliferating cells only in cardiomyocytes population. doi:10.1371/journal.pone.0009029.g004 276 transcripts showed decreased expression (Table S1 and S2). To identify the biological processes in which the genes transcriptionally affected in sik1flp/flp ES cells are involved, we performed a Gene Ontology analysis. The analysis revealed a significant enrichment in different GO Biological Process categories, such as ‘‘steroid metabolic process’’, ‘‘calcium ion transport’’, ‘‘ion transport’’ and ‘‘di-, tri-valent inorganic cation transport’’ (Table S3) in which sik1 is already known to be involved [16,22]. This observation suggests that sik1 deficiency has an effect on the transcriptome in ES cells, which is consistent with the data obtained. Sik1 is known to regulate transcription through inhibitory phosphorylation of a family of CREB coactivators, called TORCs/CRTCs, and that down-regulation of sik1 would be expected to increase the expression of CREB target genes [23] As further analysis, we decided to investigate whether genes found to be differentially expressed in the sik1flp/flp ES cells could be under the regulation of transcriptional factors belonging to the CREB family. Interestingly, Table S4 shows that CREB transcription binding sites are found to be over-represented in upregulated genes, while they are clearly under-represented in the downregulated genes. Given these data, which provide additional evidence of sik1 deficiency activity in our cells, we further analyzed the data obtained by array experiments using two stringent criteria: 1. looking for genes expressed in the heart; 2. looking for genes already known to cause cardiac phenotype when mutated (i.e. knock-out models). Using the MGI (mouse genome informatics) database, we identified 68 genes (42 downregulated and 26 upregulated) among the transcriptionally affected genes (Table S5). Interestingly, one of the most downregulated genes expressed in the heart is cdkn1c (22.75 (log2); p-value, 1.11E-16). Cdkn1c gene codifies for the p57 protein belonging to the family of KIP/CIP factors that are cyclin-dependent kinase inhibitors (CDKI) involved in cell cycle arrest. Sik1 Regulates p57kip2 Only in the Cardiogenic Program It is worth noting here that the levels of p57Kip2 are very low in proliferating cells and differentiation signals result in its accumu- lation, which is necessary to withdraw cells from the cell cycle and to move them toward differentiation [7]. Given that p57Kip2 is involved in regulating the cell cycle, it is possible to reason that it PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9029