Analysis of MMR components and MGMT in GSC and GBM cell lines. Immunoblot analysis of MLH1 (black column), MSH2 (grey column), MSH6 (white column) (Panel A) and MGMT proteins (Panel B). Bar graphs represent the mean ratios between the optical densities (O.D.) of the protein of interest and those of tubulin. The results are representative of one out of two experiments with similar results. SJ: SJGBM-2. Panel C. Analysis of MGMT activity. MGMT activity is expressed as fmol of methyl groups per mg of total protein and data are the mean (± SD) of three independent experiments. Panel D. Analysis of MGMT promoter methylation. Summary of bisulfite sequencing in 10 GSC lines. A total of 27 CpG dinucleotides (CpGs) within the promoter region of MGMT were analyzed and are represented as circles. Each row refers to one individual cell line and circle color indicates the percentage of methylation of each CpG calculated on 15 clones analyzed for each cell line. Closed circles represent fully methylated cytosines, open circles represent fully unmethylated cytosines and grey scale circles represent the indicated percentages of DNA methylation. CpGs 73, 75, 79, 80 (i.e., CpG +95, +113, +135, +137, beginning the numbering at the transcription start site and according to Everhard et al.) and CpGs 83, 86, 87, 89 correspond to those that best correlate with gene expression .