D accumulation, lobular inflammation, necrosis, and fibrosis. Intrahepatic fat was further quantified as per total pixels using the 10x objective. (French et al., 1993). Analysis of global DNA methylation levels by dot blot analyses Global DNA methylation was measured in mouse livers from each genotype and diet group using dot blot analysis as Leupeptin (hemisulfate) price previously described (Woods et al., 2012). Data are expressed according to signal intensities of 5-methylcytosine. Analysis of genomic DNA methylation levels by MethylC-seq Genomic DNA methylation according to each autosome was analyzed in three representative heterozygous liver samples from each of the three feeding groups of heterozygous mice by MethylC-seq as described (Lister et al., 2009; Schroeder et al., 2011). MethylC-seq provides a genome-wide quantitative measurement of DNA methylation that is unbiased to genomic location of CpG sites (Lister et al., 2009). Briefly, genomic DNA from each sample was sheared, annealed to methylated sequencing adapters, and bisulfite converted. DNA methylation genomic sequencing of each prepared sample was performed at the University of California Berkeley Vincent J. Coates Genomics Sequencing Laboratory. Reads were mapped to the mm9 version of the mouse genome using BS Seeker (Chen et al., 2010). CpG site methylation data in promoter and gene body regions of genes from each of 19 autosomes were combined from both DNA strands, and multiple clonal reads with the same start site were removed. Custom Perl scripts were used to convert BS Seeker output to BED files of percent methylation per CpG site. The BED files were used for data visualization on the UCSC Human Genome Browser and for further data analysis. The data were analyzed using custom Perl scripts and R programs (Schroeder et al., 2013; Schroeder et al., 2011). Quantitative PCR Liver samples from all mice were analyzed using the Vet-For-All kit and BioSprint magnetic bead extraction instrument (QIAGEN, Valencia, CA) (Osman et al., 2012). We selected a total of 21 genes involved in alcoholic liver injury and methionine metabolism pathways for expression analysis by qPCR (Bustin et al., 2009), using the geometric mean of three reference genes Hprt1, B2m, and Gapdh for normalization. Primers for each gene were designed using Primer Express and are shown in Supplemental Table 1. Data were expressed as fold changes in each feeding group of each genotype compared to the mean gene expression values in control diet samples from mice of each genotype.NIH-PA H 4065 web Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.PageImmunohistochemical staining Liver tissues from heterozygous mice were fixed in neutral-buffered formalin, embedded in paraffin, cut into 4 m thick sections, and stained with antibodies to inducible nitric oxide synthase (iNOS), DNMT1 and PPAR, each at 1/100 titer (Epitomics, Burlington, CA), followed by Donkey fluorescein isothiocyanate (FITC) labeled antibody 1/100 titer (Jackson ImmunoReaserch Labs Inc., Westgrove, PA). The expression of each of these genes had been identified by qPCR analysis as significantly altered in samples from ethanol fed mice and normalized in samples from betaine supplemented mice. Cellular fluorescence intensity was quantified in each slide in blinded fashion using a fluorescein isothiocyanate filter, Nikon morphometric software, and a Nikon 400 fluorescent microscope 40x o.D accumulation, lobular inflammation, necrosis, and fibrosis. Intrahepatic fat was further quantified as per total pixels using the 10x objective. (French et al., 1993). Analysis of global DNA methylation levels by dot blot analyses Global DNA methylation was measured in mouse livers from each genotype and diet group using dot blot analysis as previously described (Woods et al., 2012). Data are expressed according to signal intensities of 5-methylcytosine. Analysis of genomic DNA methylation levels by MethylC-seq Genomic DNA methylation according to each autosome was analyzed in three representative heterozygous liver samples from each of the three feeding groups of heterozygous mice by MethylC-seq as described (Lister et al., 2009; Schroeder et al., 2011). MethylC-seq provides a genome-wide quantitative measurement of DNA methylation that is unbiased to genomic location of CpG sites (Lister et al., 2009). Briefly, genomic DNA from each sample was sheared, annealed to methylated sequencing adapters, and bisulfite converted. DNA methylation genomic sequencing of each prepared sample was performed at the University of California Berkeley Vincent J. Coates Genomics Sequencing Laboratory. Reads were mapped to the mm9 version of the mouse genome using BS Seeker (Chen et al., 2010). CpG site methylation data in promoter and gene body regions of genes from each of 19 autosomes were combined from both DNA strands, and multiple clonal reads with the same start site were removed. Custom Perl scripts were used to convert BS Seeker output to BED files of percent methylation per CpG site. The BED files were used for data visualization on the UCSC Human Genome Browser and for further data analysis. The data were analyzed using custom Perl scripts and R programs (Schroeder et al., 2013; Schroeder et al., 2011). Quantitative PCR Liver samples from all mice were analyzed using the Vet-For-All kit and BioSprint magnetic bead extraction instrument (QIAGEN, Valencia, CA) (Osman et al., 2012). We selected a total of 21 genes involved in alcoholic liver injury and methionine metabolism pathways for expression analysis by qPCR (Bustin et al., 2009), using the geometric mean of three reference genes Hprt1, B2m, and Gapdh for normalization. Primers for each gene were designed using Primer Express and are shown in Supplemental Table 1. Data were expressed as fold changes in each feeding group of each genotype compared to the mean gene expression values in control diet samples from mice of each genotype.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.PageImmunohistochemical staining Liver tissues from heterozygous mice were fixed in neutral-buffered formalin, embedded in paraffin, cut into 4 m thick sections, and stained with antibodies to inducible nitric oxide synthase (iNOS), DNMT1 and PPAR, each at 1/100 titer (Epitomics, Burlington, CA), followed by Donkey fluorescein isothiocyanate (FITC) labeled antibody 1/100 titer (Jackson ImmunoReaserch Labs Inc., Westgrove, PA). The expression of each of these genes had been identified by qPCR analysis as significantly altered in samples from ethanol fed mice and normalized in samples from betaine supplemented mice. Cellular fluorescence intensity was quantified in each slide in blinded fashion using a fluorescein isothiocyanate filter, Nikon morphometric software, and a Nikon 400 fluorescent microscope 40x o.
