Fig. 1

Analysis of CpG methylation of PCDBH15 in melanoma cell lines and patient samples and re-expression after 5azadC treatment of WM266-4 cell line. A The percentage of DNA methylation of each CpG in PCDHB15 was analyzed by bisulfite conversion followed by pyrosequencing of the CpGs indicated as black dots. CpGs on the sequence but not amplified in pyrosequencing are indicated as dotted lines. The CpGs of the Illumina 450 K array are indicated by an asterisk: for PCDHB15, cg27328673, cg23974473 and cg09135656 at + 566, + 610 and + 664pb from the TSS, respectively. B–C The DNA methylation mean level of PCDHB15 (B) was measured in two pairs of cell lines originating from two different patients, WM115/WM266-4 cells and WM983A/WM983B (B), as well as in genomic DNA obtained from 27 patient samples, primary (n = 18) or metastases (n = 9) (C). Data are presented as box plot of the median DNA methylation percentage of CpGs in black (6 CpGs for PCDHB15). The median values for primary and metastasis samples are 61.5% and 71.6%, respectively, Jarque–Bera’s test to analyze normality, Fisher’s test to analyze variances and Student t test were performed, n.s = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. D Normalized beta values for the Illumina probes of DNA methylation for primary melanoma (PRM), lymph node metastasis (LNM) and distant organ metastasis (DOM) from TCGA-SKCM DNA methylation data of PCDHB15 were summarized as a heatmap. A violin plot was used to highlight CpGs identified as hypermethylated in metastatic cell lines in our previous study. Differential DNA methylation was assessed by the Wilcoxon rank-sum test. All p values from multiple comparisons (> 50 tests) were corrected using the false discovery rate (FDR) method. * = p < 0.05. Transcription factor clusters from transcription factor ChIP-seq clusters (340 factors, 129 cell types) from ENCODE 3 were indicated as black/grayscale. WM266-4 cells were treated with increasing concentrations of 5azadC daily during 72 h (d1, d2, d3). E At day 4, DNA methylation of PCDHB15 at exon 1 was measured by pyrosequencing (n = 2 for WM266-4 and WM115 cells; n = 3 for 5azadC-treated cells). The box plots show the percentage of DNA methylation of the analyzed CpGs (from panel A). F The mRNA quantification of PCDHB15 by RT-qPCR was performed at day 7, using the TBP gene as reference gene and normalized according to the expression level found in the WM266-4 cells (n = 4, SEM are shown). Fisher’s test to analyze variances and Student t test were performed, ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001