Fig. 4

Alterations in DNA methylation but not hydroxymethylation in EtOH-exposed primary neuroepithelial stem cell cultures. a Stable levels of DNA methylation within the differentially methylated regions of the imprinted genes Snrpn and Peg3. Stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Genomic DNA was collected at days-3 and 7, and analyzed for alterations in DNA 5mC and 5hmC using glucMS-qPCR. Graphs represent three independent biological replicates (N = 3), with three qPCR measurements each. b Quantification of Snrpn transcript levels using RT-qPCR. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. Differences were measured using a one-way ANOVA, error bars represent SEM. c, d Measurement of 5mC and 5hmC within the regulatory regions of eight genes identified in previous studies of DNA hydroxymethylation. Cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Genomic DNA was collected at days-3 and 7, and analyzed for alterations in DNA c 5mC and d 5hmC using glucMS-qPCR. Differences were measured using a one-way ANOVA, error bars represent SEM. Graphs represent three independent biological replicates (N = 3), with three qPCR measurements each