7Aberrant methylation of promoter region mediated the silencing of MAL in HNSCC. ( A) Pharmacogenetic treatment of nine HNSCC cell lines with or without 5-Aza-dC and/or TSA. Primary keratinocytes were used as the positive control (POS.), and no template added in the PCR reaction was regarded as the negative control (NEG.). (B) Schematic representations of the location of the CpG islands within the promoter and the intragenic regions of MAL and the location of the bisulfate-based sequencing within the CpG island. (C) Representative results from the bisulfate-treated genomic DNA sequencing to detect the methylation status of the BS-1 and BS-2 regions in two representative HNSCC cell lines and seven HNSCCs as compared with the methylation level in primary keratinocytes and matched normal epithelium cells (P < 0.05). The numbers indicate the CG dinucleotide within the CpG island in the promoter. Black and white circles represent methylated and unmethylated CpGs, respectively.