2001; Ikonomidou et al., 2000; Kilburn et al., 2006), we also examined EtOH's effects on the levels of an early apoptosis marker Annexin V and found noticeable increases in Annexin V immunoreactivity in hESCs after 20 mM EtOH treatment (Fig. S3A). Based on these immunohistochemical data, we next examined EtOH's effects on the expression of 6 stemness marker genes by quantitative RT-PCR (Fig. S3A). Treatment with EtOH (20 or 50 mM) resulted in significant downregulation of DNMT3B, NANOG, OCT4 and SOX2, with the remaining FOXD3 and TERT also showing a trend of decreased expression (Fig. S3B). We then reasoned that if pluripotency markers that maintain self-renewing hESCs were downregulated by EtOH, this is likely to have significant effects on the regulation of “downstream” differentiation markers. Analysis of qRT-PCR data for a set of previously described differentiation markers representative of the mesoderm (BRACHYURY, HAND1, MEOX1), definitive and primitive endoderm (CDX2, EOMES, GATA4, GATA6, SOX7, SOX17, TBX6) and ectoderm (NESTIN, PAX6), respectively (Teo et al., 2011) revealed significant up- or down-regulation of 9 out of these 12 markers with EtOH treatment (Fig. S3C). Based on the above data and the profound effects of EtOH exposure during the blastocyst stage on subsequent embryo
