DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases (Acil, Pvul and BsaAl). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion (AciI-active 50 + 176 bp; PvuI-active 37 + 180 bp; BsaAI-active 192 + 25 bp). (B) COBRA with each of the three different endonucleases (Acil, Pvul, BsaAl) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ. (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.