Figure 1

Effect of zebularine on mESC differentiation. (a) Detection and comparison of cardiac markers by RT-PCR in Control, AzadC, NO and zebularine treatment, undifferentiation markers are less detected and cardiac markers showed an enhanced expression after treatments. (b) Time course of zebularine treatment on lineage formation: mESCs cells were cultured as hanging drop during 3 days to form EBs and differentiate to derivatives of the three germ layers and then leaved in culture without treatment (Control) or treated with 30 μM zebularine. Gene expression was assessed in parallel at differentiating days 3, 5 and 7. Cells cultured without LIF (−LIF) are used as negative control for lineage formation. RT-PCR was performed with primers to detect pluripotency markers (Oct3/4 and Nanog), endoderm-specific markers (Sox17, Pdx1, FoxA2, Pax4, P48, Mist and Nkx6.1), mesoderm-specific markers (Brachyury, Gata4, Nkx2.5, αSMA, SM22α, Desmin, Flk1, Hrt1, Mef2c, tbx5, BMP2, BMP4, Serca2 and Noggin) and ectoderm-specific markers (Wt1, Snail1, Slug, Otx2, Zic1, Nestin, Ngn, Pax6. Islet1 and Sox1). β-Actin was used as the input control. (c) Apoptosis comparison for control-, zebularine- and AzadC-treated EBs. Time-course analysis and comparison of Annexin-V binding at differentiating days 3, 5 and 7, indicating that cells treated with zebularine are not undergoing apoptosis. Values were normalized from the means of the controls. Data are representative of three independent experiments statistically analyzed with analysis of variance (ANOVA) I (*P<0.05, **P<0.01 and ***P<0.001). Different letters at the top of the bars represent statistically different values; same letters represent statistically same values in discriminatory analysis Tukey’s B test