Knockdown of Sirt2 results in the alteration of mouse ESC lineage commitment.

<p>(<b>A</b>) Phase-contrast images of EB at day 6. All Figures 100×. (<b>B</b>) Real-time PCR shows the relative mRNA expression levels of the ectoderm marker genes (Otx2, Sox1 and Pax6), the mesoderm marker genes (Gata6, Mesp2, Mixl1 and T) and the endoderm marker genes (Cxcr4, Gata4, LaminB and Sox17) in control (shpLKO.1) and two Sirt2 stable knockdown cell lines (shSirt2-1 and shSirt2-2) during EB differentiation at day 6. Data were normalized to <i>Gapdh</i> mRNA expression levels and are means ±SEM (n = 3). <i>P</i><0.05(*), <i>P</i><0.01(**) and <i>P</i><0.001(***) vs. control EBs generated from shpLKO.1-infected mouse ESCs (One-way ANOVA). (<b>C</b>) Western blots to analyze the protein levels of Tuj1, Actin (α-SMA) and Gata4 in EBs generated from control and Sirt2 knockdown ESCs at day 9. GAPDH was used as a protein loading control. (<b>D</b>) Immunostaining for Tuj1, Actin (α-SMA) and Gata4 in EBs generated from control and Sirt2 knockdown ESCs at day 9. Cells were counterstained with DAPI (blue). All Figures 100×.