software (ASI). The chromosomes were counterstained with 4,6- diamino-2-phenylindole (DAPI) in an anti-fade solution (ASI). Twenty metaphases were evaluated per cell line examined. Statistical significance (P,0.05) was calculated by the Kruskal- Wallis test followed by the post-hoc Dunn’s. All statistics were carried out using GraphPad Prism software version 4 for Windows (GraphPad). Treatment with Epigenetic Chemical Inhibitors To analyze the DNA demethylation effects induced by 5-aza-29- deoxycytidine (5AzaCdR; Calbiochem, Merck, Darmstadt, Ger- many) on a genome-wide scale and in specific genes, mouse cell lines (melan-a, 4C, 4C112 and 4C11+) in log phase were incubated in medium supplemented with 10 mM of the chemical compound for 48 hours. To analyze the histone deacetylase effects induced by Trichostatin A (TSA; Calbiochem) on gene-specific expression, mouse cell lines were incubated in medium supple- mented with 40 nM of the chemical compound for 16 hours. Finally, to analyze the synergistic effects between 5AzaCdR and TSA on gene-specific expression, mouse and human cell lines were incubated in medium supplemented with the both compounds, at the concentrations and time indicated. In all of them, inhibitor- treated cell lines were compared against their control cell counterparts which were cultured in a paired way but in inhibitor(s)-free culture medium. These treatment conditions do not significantly induce cell toxicity since they were not able to impair cell proliferation, as previously determined by us after performing methyl thiazol tetrazolium (MTT) assays [11]. In addition, Trypan Blue stain 0.4% was used to monitor cell viability in a Countess Automated Cell Counter (Invitrogen, Carlsbad, CA). Digestion of Genomic DNA with MspI and HpaII To evaluate the relative amount of global DNA methylation, 2 mg of genomic DNA extracted from melan-a, 4C, 4C112 and 4C11+ monolayers, as well as their 5AzaCdR-treated counterpart cells, were digested or not with 2 mL of enzymes MspI or HpaII in separeted reactions (Fast Digest, Fermentas, MD). The DNA was digested for 16 hours at 37uC. Afterwards, 1 mL of each enzyme was added, and the reactions were maintained for 1 hour at 37uC. Then, samples were resolved onto 0.8% agarose gel electropho- resis. The intensity of the bands corresponding to intact genomic DNA, genomic DNA digested with MspI, and genomic DNA digested with HpaII in each sample was determined using the ImageJ software. Percentage of methylation was measured as follows: relative global DNA methylation content = (HpaII-MspI) 6100/ genomic DNA. Percentage of demethylation was calculat- ed as follows: 100-percentage of methylation. RNA Extraction and Microarray-Based Gene Expression Studies Mouse cell lines (melan-a, 4C, 4C112 and 4C11+) were grown and treated as previously described. For DNA demethylation and high-throughput microarray-based gene expression studies, cells were harvested by trypsinization at 48 hours. Total cell RNA which was extracted from 5AzaCdR-treated and control cell monolayers using TRIzol reagent (Invitrogen), was purified using RNEasy MinElute Cleanup spin columns (QIAGEN, Dusseldorf, Germany), according to manufacturer’s instructions. Only RNA samples with an absorbance A260/A280 nm ratio greater than 2.0 were used for microarray hybridization. Briefly, 2 mg from total RNA were amplified by an in vitro transcription reaction and biotin-labeled. 39-IVT Expression Arrays (GeneChip Mouse Novel Candidate Biomarkers in Melanomas Genome 430_2.0, format 49; Affymetrix Inc., Santa Clara, CA) which were hybridized under standard conditions for 16 hours at 45uC and 60 rpm, were washed, stained with streptavidin- phycoerythrin and scanned using an Affymetrix GeneChip Scanner 3000 7G. Gene expression experiments using Mouse 430_2.0 Arrays were done following the Affymetrix Expression Analysis Technical Manual. Hybridization reactions and scanning were carried out at Associac¸ a˜o Fundo de Incentivo a` Psicofarma- cologia (AFIP, Brazil). Microarray and Pathway Analyses In the chosen arrays, there are 45,101 probe sets which are designed to investigate approximately 34,000 mouse transcripts. We used GCOS software version 1.4 (Affymetrix) to extract expression values (raw array data) generated from Mouse 430_2.0 Array experiments, according to standard procedures by Affyme- trix Systems. To identify differentially expressed genes in the transitions from melan-a to 4C, 4C to 4C112, and 4C112 to 4C11+ cell lines, class-comparison experiments were performed, and transcripts were selected as statistically significant by the two- class unpaired SAM analysis with 700 random permutations (FDR,0.05). The experiments were carried out in three biological replicates. To estimate differences on gene responsiveness upon 5AzaCdR treatment in each cell line, 5AzaCdR-treated cells were compared against their previously untreated counterpart cells. Transcripts were selected as statistically significant by the two-class paired SAM analysis with 500 random permutations (FDR,0.05). The experiments were carried out in two biological replicates. The raw array data (.CEL files) were first quantile normalized with background correction using RMAExpress version 1.0.5 [54]. Next, the normalized expression values (log2 scale) were filtered due to strict quality criteria. Filtering and statistics were carried out using MultiExperiment Viewer (MeV) version 4.6 from The Institute of Genomic Research (TIGR; Rockville, MD) [55]. This filtering reduces the number of probes to 36,080 but increases the consistency of the measurements as shown by the removal of genes that did not vary much in expression over the conditions of the experiment. This variance filter was performed after quality control filters, such as lower and percentage cutoffs, were imposed. This methodology insures that the genes that were checked for variance also contained a level of quality data. Afterwards, genes showing at least a two-fold enrichment and a median false discovery rate (FDR) and a Q-value threshold of 5% after SAM statistical analysis [56], were chosen. For all comparisons, we removed probes that corresponded to hypo- thetic proteins, riken cDNAs, cDNA sequences and transcribed loci because all of them are in silico predicted or not well characterized just yet. This approach yielded a more accurate identification of differences on global gene expression profiles, making the final data sets more reliable for future validation. The differentially expressed genes identified here were submitted to an unsupervised hierarchical clustering analysis (HCL) [57] using the average linkage algorithm and the Pearson correlation metric distance to arrange genes and samples according to similarity in patterns of gene expression. Pathway analysis was generated by using the PANTHER software. The binomial test was applied to determine whether there was a statistical representation of genes (P,0.05) [58]. Accession Numbers The raw microarray data are accessible from the ArrayExpress (E-MEXP-2512 and E-MEXP-2517). PLOS ONE | www.plosone.org 16 September 2012 | Volume 7 | Issue 9 | e44800