Sik1 Controls Cardiomyogenesis Figure 3. sik1 deficiency affects terminal cardiogenic differentiation. (A) Q-PCR of a pluripotency gene (Oct3/4), of an early mesodermal marker (brachyury), of pre-cardiac mesodermal markers (mesp1, mesp2, Isl1), early cardiogenic markers (Mef2c, Tbx5, Nkx2.5) and terminal cardiac markers (a-MyHC, Mlc2V, aCach and cTnI) during in vitro differentiation of wild-type and sik1flpflp ES cells. (B) Q-PCR on hematopoietic/endothelial markers (runx1, vegfC and flk1). n = 3, *, P,0.05, **, P,0.001 vs. control cells. P values were calculated using a two-tailed, unpaired t test. doi:10.1371/journal.pone.0009029.g003 the wild-type and sik1flp/flp populations, thus indicating that the EBs carrying the sik1 mutant alleles have the same competence to acquire the neural phenotype as the wild-type EBs; supporting the idea of a cardiac specific effect of sik1 deficiency. The p57Kip2 Transcript Is Strongly Downregulated in sik1flp/flp ES Cells Given the role of sik1 as transcription regulator [12], we studied the transcriptome influence of sik1 in ES cells. We decided to perform the experiment on undifferentiated ES cells, comparing wild-type and sik1flp/flp cells, for three reasons: 1. to consider a homogenous cell population compared to differentiating EBs, which are known to contain different cell types; 2. to avoid an enrichment of cardiac specific genes, already found to be significantly altered in the previous analysis, and 3. to possibly reveal an early transcriptional event that may play a role in embryonic development. Using the filtering criteria of a two-or-greater-fold change in expression and a false discovery rate of less than 5%, 448 out of over 22,000 transcripts were differentially expressed. Under these conditions, there were 172 transcripts with increased expression in the sik1flp/flp ES cells, while PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9029