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 with promoter methylation in the middle panel of Fig. 5) were definitively validated. C2CD2L, GUSBP1 and TMEM217 were significantly upregulated and demonstrated concentration dependency to EtOH treatment (Fig. 6A). By contrast, P2RX3, PIK3C3, SLC12A4 and SLC25A30 were significantly downregulated (Fig. 6B). Thus, most of the genes subjected to qRT-PCR analysis showed the expected trends of gene expression. Of the downregulated genes, SLC12A4 is involved in K-Cl transport and participates in cell volume homeostasis and cholesterol metabolism (Adragna et al., 2004; Zhou et al., 2004). The gene is localized to chromosome 16, which we identified as one of the hotspots, (see Fig. 3). P2RX3 codes for a ligand-gated cation channel, which is involved responses to extracellular ATP, cation transport, Ca-mediated signal transduction, and behavioral responses to pain (Burnstock, 2007). However, the specific functional role of these genes in EtOH-induced hESC self-renewal and potency remain to be investigated.
