A genome-wide microarray (Affymetrix Human Genome U133 Plus 2.0 Array platform) was used to generate gene expression data for hESCs in order to compare the concentration-dependent effects of EtOH (0, 20 or 50 mM) on two different hESC lines, H1 and H9 (Fig. 1). The H1 and H9 cell lines were derived from male and female fetuses, respectively (Thomson et al., 1998). To find co-expression modules related to EtOH treatment and differentiation status we used the Weighted Gene Co-Expression Network Analysis (WGCNA) (Fig. 1A) (Langfelder and Horvath, 2008; Zhang and Horvath, 2005). We also examined the molecular signatures of differentiation from undifferentiated stem cells to embryonic bodies (EBs) in the absence of EtOH treatment. Comparisons of H1 and H9 heatmaps from representative modules are shown (Fig. 1B). We selected three representative modules that showed the same pattern of expression changes after EtOH treatment in both H1 and H9 hESCs. This is to demonstrate an example of EtOH-induced alterations in gene signatures independent of cell lines. Volcano plots (Fig. 1D) demonstrated similarities in EtOH's effects on expression of genes in the
