In summary, we have used a multiplatform integrative approach to identify a short list of robust methylated genes in melanoma. We confirm previous reports that COL1A2, THBS1, TNFRSF10D and UCHL1 are highly methylated in melanoma, thus providing further evidence that these genes are highly important in melanocytic neoplasia. Others have used somewhat similar integrative approaches [59,60]. Sjaputera et al. [60] compared methylation array data with expression data with an arbitrary methylation cut off; and Loss et al. [59] integrated methylation and expression data followed by logistic regression to identify the most significantly affected genes. However, they did not include re-expression data following 5azadC treatment, or quantification of the degree of methylation using the Epityper or a similar assay. While these other two studies were done on different cancer types (breast cancer and lymphomas), one common methylated gene, COL1A2 was identified. Taken together, this suggests that COL1A2 is a gene for which methylation is more generally associated with tumorigenesis across different cancer types. Materials and Methods Cell Culture A panel of 12 melanoma cell lines derived from primary cutaneous melanomas or their metastases were used [14]. All cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum as described previously [19,61]. Primary human melanocytes were obtained from neonatal foreskins and cultured in 10% heat-inactivated fetal calf serum (CSL, Melbourne, Australia) in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 3 mM HEPES with the addition of 6 ng/ml cholera toxin and 16.2 nM phorbol 12- myristate 13-acetate (PMA) (Sigma Chemical Co., St. Louis, MO) as previously described [62]. All tissue was taken with written informed consent under a protocol approved by the Queensland Institute of Medical Research Human Research Ethics Committee (HREC), approval number H0311-084 (P726). The 2 colorectal cancer cell lines Co115 and LIM 2405, esophageal cancer cell lines OE19 and OE33, glioma cell lines T46 and T50 were purchased from the American Type Culture Collection (http://www.atcc.org/). DNA extraction, Bisulfite Conversion and Illumina Infinium Methylation27 Array Hybridization QIAGEN DNeasy Blood and Tissue Kits were used to isolate genomic DNA from cells in log phase growth as per the manufacturer’s instructions (QIAGEN,Hilden). All samples were run on an Agilent Bioanalyzer (Agilent, CA, USA) using a DNA 12000 LabChip kit to check for DNA integrity, purity and concentration. 500 ng of genomic DNA from 11 melanoma cell lines and a reference pool of melanocytes derived from several donors were bisulfite treated using an EZ-96 DNA methylation kit (Zymo Research, CA, USA) as per the manufacturer’s instructions. They were then hybridized to Infinium Methylation BeadChips (Illumina,CA,USA) containing 27,578 CpG loci covering more than 14,000 genes [17]. All reagents and procedures for washing, detection and scanning were performed according to the BeadStation 5006 system protocols (Illumina, CA, USA). Beadarray methylation analysis Percent methylation (beta) was calculated from the ratio of fluorescent signal intensities of the methylated (M) and unmethy- lated (U) alleles, for each sample at each specific CpG site, using the equation beta = Max(M,0)/[Max(M,0)+Max(U,0)+0]6100. Epigenetic Gene Silencing in Human Melanoma On this scale unmethylated sites are represented by beta values close to zero, while heavily methylated sites show values approaching 100%. We then expressed each site specific melanoma cell line methylation value as a delta difference, compared to that of melanocytes. In this way negative values represented a cell line and site specific decrease in methylation, while positive scores indicated a relative increase in the degree of methylation. The provided manifest file linked individual meth- ylation sites to official gene symbols, which we used to associate the methylation data to that of mRNA expression and demethylation, as described below. The data are MIAME compliant and have been deposited in Gene Expression Omnibus database (http:// www.ncbi.nlm.nih.gov/geo/) under accession number GSE28356. Microarray gene expression profiling These data (GSE7127) were generated as part of previously published studies [18,19]. Briefly, 5 mg of total RNA from 35 melanoma cell lines and one melanocyte foreskin pool were applied to Eukaryotic One-cycle Target Labelling and Control Reagents kits, according to the manufacturer’s instructions (Affymetrix, CA, USA), and 15 mg of the resulting fragmented cRNA mixtures was then hybridized to an Affymetrix Human Genome U133 Plus 2.0 Array for 16 hr at 45uC. Chips were then washed and streptavidin phycoerythrin post-stained, before scanning on an Affymetrix GeneChip Scanner 3000. Relative expression values for each probe set were generated from the raw image data files using the Affymetrix PLIER algorithm in ArrayAssist 1 Version 4.20 (Stratagene). The resulting data were imported into GeneSpring GX v7.3 (Agilent Technologies) where data values less than 0.1 were set to 0.1 prior to log transformed to base 2. The expression values were then centrally normalized to the median expression value for each sample and the median expression value for each probe set. For the current study, expression data for each of the 11 melanoma cell lines for which we have Illumina Infinium Methylation Array were expressed as a ratio, per gene symbol, compared to that of the included melanocyte cell line. In this way we are able to match a single methylation value to an expression estimate for each of the 11 cell lines with matched data. 5AzadC demethylation profiling These data were generated as part of a previously published study [14] (GSE32492). Briefly, cells were split to 20% confluence 24 hr prior to commencing a 3-day treatment with either 5 mM of 5AzadC (Sigma) from 100 mM 50% acetic acid dissolved stock, or mock treated with the same volume of phosphate buffered saline (PBS)/50% acetic acid. The 3-day incubation was followed by a 4- hr incubation with 300 nM TSA (Sigma) prior to total RNA extraction from cells in log phase growth (RNeasy Midi-kits – Qiagen, Hilden) and on-column DNase digestion. Samples with an Agilent Bioanalyzer (Agilent, CA, USA) determined RNA integrity number (RIN) of .8.0 were used for microarray analysis. Biotinylated cRNA were prepared from 500 ng of total RNA using an Illumina TotalPrep RNA Amplification Kit (Ambion, TX, USA) and 1500 ng was hybridized to Sentrix Human-6 Expression version 2 BeadChips (Illumina, CA, USA) prior to washing, detection, and scanning according to the BeadStation 500GX system protocols (Illumina). Expression profiles generated for each cell line before and after drug treatment (expressed as a fold-change ratio) showed that across the panel of 12 cell lines a total of 8,144 non-redundant genes were re-expressed with .2-fold change after treatment (between 1,457 and 3,386 genes in individual samples). Genes reactivated in all 12 cell lines were removed from further analysis PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26121