Figure 2.

Zebularine promotes an overexpression of cardiac markers. (a) Timeline approach for differentiation of mESCs, showing the time periods for EBs formation, EBs adhesion, untreated control cells (no addition) and zebularine application. (b) Real-time PCR showing an overexpression of genes essential for cardiac development and function after zebularine treatment. The histograms represented the fold increase of each gene after treatment compared with the basal level of untreated control cells. Results are means±S.D. for three different experiments. (c) Western blot detection of cardiac protein expression; β-actin was used as a loading control. (d) Immunodetection corroborated the cardiac lineage markers after zebularine treatment; nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars; 50 μm. (e) Results from flow cytometric analysis showing the percentage of cells expressing cardiac-specific proteins in control and zebularine conditions. Data are represented as mean±standard error (±S.E.) for three different experiments. Statistical significance (*P<0.05) was determined by t-test