We next validated genes identified from the combined analysis. Based on the observed decreases in stemness markers (Fig. S3), we continued to focus on molecular signatures affected by EtOH-induced DNA methylation changes that may have significant roles in stem cell maintenance and differentiation process. Therefore, we examined EtOH's effect on the subset of genes that are potentially differentiation-related as shown in Fig. 5. DAVID analysis on the identified top 30 genes was performed to examine their functional implication. We found these genes were mostly associated with neuroactive ligand-receptor interaction, vascular smooth muscle contraction, calcium signaling pathways, and energy metabolism. Energy metabolism and its associated pathways are considered to be important cellular processes in maintaining stem cell self-renewal and potency (Cho et al., 2006; Rafalski et al., 2012; Ramm Sander et al., 2013). For validation, the levels of mRNA for selected genes were assessed by qRT-PCR analysis (Fig. 6). Even though we relied on GAPDH as a single reference gene (Carnahan et al., 2013), we found that most of the genes in our experimental set (subset of EtOH-induced differentiation related genes
