since they are likely to represent cellular stress response to 5AzadC treatment, or promoter demethylation of genes normally silenced within the melanocytic lineage.This filtering left 3125 genes to be considered from cross-analysis with the Beadarray27 methylation and U133 Plus 2.0 mRNA expression data described above. Combining mRNA profiling, demethylation and Beadarray27 data across 11 melanoma cell lines Fold-change expression profiling data (compared to melanocyte pool), fold-change demethylation data (compared to matching untreated cell line) and Illumina methylation profiling data (delta % methylation compared to the melanocyte pool) for each of the 11 cell lines for which all data were available, were imported into Microsoft Excel and linked via official gene symbol (HUGO Gene Nomenclature Committee). Data filters were then applied to each of the three data types in order to identify genes with clear evidence of methylation (through both the presence of increased methylation values, and with increased expression following demethylation) and evidence of reduced expression compared to the melanocyte pool. Genes symbols were filtered to identify those in which at least 2 samples showed $60% methylation (Beadarray27) correlated to an average post-demethylation re- expression fold-change of .4 and 4-fold mRNA global decrease across the panel of 11 melanoma lines. From this set of 26 genes, after removing oncogenes (ADM, ENPP2, RAC2, SERPINE1), genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as ‘‘orf’’ (C10orf116, FLJ20551, FLJ20647), and the presence of false positives on the Infinium Methylation chips (i.e. multiple instances where cell lines were methylated on the Beadarray27 but showed no re-expression with 5AzadC treatment; EEF1A2, HSPA2), 16 genes remained for initial follow up (COL1A2, CRABP2, CRIP1, FAM46B, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D, UCHL1, ALDOC, COL12A1, GALM, VAMP5 and VAMP8). Of these, 12 genes (COL1A2, CRABP2, CRIP1, FAM46B, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D, UCHL1 and VAMP8) were subject to further validation as described below. Quantitative RT–PCR mRNA extraction & expression array data were obtained as previously described [14,18,19]. To confirm the validity of the microarray expression data, the mRNA levels were assessed by quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR) (see Table S2 for primer sequences) in the 5 cell lines with the highest expression differences before and after 5AzadC treatment. First-strand cDNA synthesis was performed with 3 mg total RNA for each sample in a total volume of 20 ml using Superscript III reverse transcriptase and random primers (Invitrogen, CA, USA). Subsequent PCR reactions were carried out on a Corbett RotorGene 6000 (Corbett Research, Australia) using SYBR Green RT–PCR Master Mix (Applied Biosystems, Foster City, CA). CLTA (clathrin light chain mRNA) was chosen as the normalization control transcript based on minimum variation across the cell lines as assessed by microarray [14]. EPITYPER Assay The Sequenom EpiTYPER assay is based on in-vitro transcription and base-specific cleavage of a PCR amplicon and the subsequent analysis of the resulting RNA fragments by MALDI-TOF mass spectrometry [15,20]. EZ-96 DNA methylation kits (Zymo Research, CA, USA) were used for bisulfite treatment of 1 mg of genomic DNA from 44 melanoma cell lines, 1 nevus cell line, 2 colorectal cancer cell Epigenetic Gene Silencing in Human Melanoma lines (Co115 and LIM 2405), 2 esophageal cancer cell lines (OE19 and OE33), 2 glioma cell lines (T46 and T50), and 30 fresh-frozen melanoma tumors. DNA from pools of melanocytes was used as reference. Each gene promoter was divided into several amplicons (Table S1). The target regions were then amplified using the primer pairs containing a T7-promoter tag (forward: 59-AGGAAGAGAG-fw primer-39, reverse: 59-CAG- TAATACGACTCACTATAGGGAGAAGGCT-rev primer-39) to allow further in vitro transcription. One microliter of modified DNA was used for the PCR reactions carried out in a total volume of 5 ml. Unincorporated dNTPs were dephosphorylated by incubation at 37uC for 40 min in the presence of shrimp alkaline phosphatase (SAP) (Sequenom). Two microliters of this SAP-treated PCR mixture were used as a template in a 7 ml transcription reaction containing RNase A and T7 polymerase (Sequenom). Transcription and digestion were performed simul- taneously at 37uC for 3 h. After the addition of 20 ml of H2O and 6 mg of CLEAN resin (Sequenom), 22 nl of the cleavage reactions were dispensed onto silicon chips preloaded with matrix (SpectroCHIPS, Sequenom). Mass spectra were collected using a MassARRAY mass spectrometer (Bruker-Sequenom) and analysed using proprietary peak picking and signal-to-noise calculations (Sequenom Epityper v1.0.5). The relative amount of methylation (% methylation) was determined by comparing the signal intensities between the mass signals of methylated and non-methylated template. For data analysis only unique CpG units (units can contain one or more consecutive CpG dinucleotides) are included. CpG units overlapping with other cleavage fragments in the mass spectrum were excluded from data analysis. CpG methylation ratios were filtered using an uncertainty threshold of 10%. Only data values (CpG methylation ratios) with an estimated error smaller than 10% were included in the analysis. This filtering ensured that only precise data values were used for downstream calculation. For each gene, the amplicons presenting no significant difference in methylation between melanoma cell lines and melanocytes were dropped from the analysis (Figure S2). In each amplicon, the informative CpG sites were then scored defining the CpG island for each gene. Only the CpG sites presenting high methylation levels in melanoma cell lines were included in the final percentage of methylation. The average % of methylation for each gene was then defined as the average value across the melanoma cell line panel which was compared to melanocytes. Statistical Analysis Spearman test was applied for the correlation between mRNA expression and methylation of the genomic region assessed. A t- test (t = r/Sr) was performed to obtain the significance (Sr = (12r2)/n) of the Spearman coefficient. Western blot analysis Total cell lysates from 1.107 cells were generated as previously described [63]. Samples (30 mg protein) were resolved by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Antibodies raised against the following proteins were used for Western blotting: anti-THBS1 (ab88529, 1 in 1,000 dilution; Abcam, Cambridge, UK), anti-UCHL1, (1 in 1,000 dilution; Sigma Prestige antibodies, St. Louis, MO), and anti-GAPD (1 in 5,000 dilution; R&D Systems, Minneapolis, MN). Detection was performed using the appropriate peroxidase- conjugated secondary antibody and a Western Lightning chemiluminescent reagent plus kit (PerkinElmer LAS, Inc., Boston, MA). PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26121