Figure 4

The effect of chronic ethanol exposure on murine embryonic fibroblasts (MEFs). (a) MEFs were exposed to ethanol for a period of 7 days at which time the methylation status of individual Plk CpG islands was determined by MSP analysis. U = unmethylated, M = methylated. (b) Plk4 and Plk1 transcript levels in Plk4 wild type (Plk4 ^+/+) MEFs were determined after 7 days of ethanol exposure by qPCR. RQ values were normalized to the level of transcript found in untreated control MEFs. Standard error was calculated based on the minimum and maximum values from the mean expression levels (RQ) (c) Immunofluorescence analysis Plk4^+/+ MEFs exposed to 25 mM and 50 mM ethanol for a period of 7 days. Centrosomes were detected by γ-tubulin staining and DNA by Hoechst staining. A graphical representation of cells exhibiting multiple centrosomes and multinucleation is underneath. Shown are the results of three independent experiments in which more than 200 cells were analyzed each time for each condition. Error bars indicate standard error. (d) Global methylation analysis of genomic DNA from MEFs after 7 days alcohol exposure as determined by MSP analysis of B1 elements. The error bars represent the upper and lower limit of the standard error from the mean. (e) Plk4 transcript levels as determined by qPCR of MEFs exposed to ethanol for 7 days in the presence of 5-aza-2'deoxycytidine (AZA), trichostatinA (TSA) and valproic acid (VPA). RQ values were normalized to the level of Plk4 transcript in untreated control MEFs. The error bars represent the upper and lower limit of the standard error from the mean expression level (RQ)