Figure 4

Identification of methylation sites at p53 binding sequence of IGFBP-3 promoter. (a) BSP in P0 and P4. Forty CpG sites were shown and each circle represents a CpG dinucleotide. Open circle represents non-methylated CpG dinucleotude and black circle represents methylated CpG dinucleotide. The enclosed boxes represent CpG dinucleotide located in the p53 consensus binding site. Seven clones were sequenced in each cell line. (b) The methylation frequency in P0 and P4 from -210 to -179 regions of IGFBP-3 promoter. (c) Schematic diagram of designed constructs for site-directed mutagenesis assay. The wild type construct contains IGFBP-3 promoter region from --253 ~+61. The mutant sequences carry the same region but with point mutations: (-210, -206) represent as Mut A constructs, and (-183, -179) represent as Mut B constructs. Mut A+B construct contains all four nucleotide mutation. (d) Luciferase activities of 293T and P4 transfected with wild/mutant types of IGFBP-3 promoter constructs. Luciferase activity was normalized against renilla activity. Transfection with pGL3-basic vector (pGL3) was used as negative control. Transfections were carried out in triplicate and were done in at least three independent experiments.