Gene expression analysis of WT (SIRT1^+/+) and KO (SIRT1^−/−) EBs by real-time PCR analysis. (A) Quantitative RT-PCR (qRT-PCR) analysis of stem cell marker Oct4 and Nanog mRNA levels in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (B) qRT-PCR analysis of mRNA levels of epiblast marker Fgf5, mesoderm markers T, Wnt3, and transcription factor Tal1/Scl in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (C) qRT-PCR analysis of mRNA levels of trophectoderm marker Cdx2, neuroectoderm marker Sox1, and cardiac lineage marker Nkx2-5 in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (D) qRT-PCR analysis of mRNA levels of endoderm/mesoderm markers GATA-4, GATA6, and Sox17 in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. Day (D) 0 represents undifferentiated ESCs. Graphs in panels A through D were plotted in logarithmic scale. Data are relative to SIRT1^+/+ D0 control and mRNA levels of each gene were compared between SIRT1^+/+ and SIRT1^−/− EBs at each time point. Results were the average of 3 independent experiments, each performed in triplicate. *P < .05.