Figure 2

MCT-1 promoter associates with p53. (A) Six putative p53-binding sites on the MCT-1 promoter region are illustrated. The primers for ChIP assays are indicated by 166, 173 and 199 bp on the MCT-1 promoter region with the horizontal arrows. (B) Following the genotoxic stress, MCF-10A genomic DNA was immunoprecipitated (IP) with p53 and RNA polymerase II Ab. The original input and the Ab-IP complexes were amplified by a conventional PCR analysis with the primers for MCT-1 promoter, MCT-1 coding region, or GAPDH gene. MCT-1 promoter DNA was identified by primers specific for the 166, 173, and 199 bp fragments in the IP complex of p53 Ab (lanes 6, 8, 10) but not normal IgG (lane 4). MCT-1 coding region was undetectable in the p53-IP complex (lane 3). The GAPDH gene interacting with RNA polymerase II was recognized as a positive control (lane 1). (C) In quantitative PCR (q-PCR) comparison, the relative values were normalized with the original input and then compared with the non-specific MCT-1 coding region (NS site). (D) MCT-1 promoter DNA fragment (166 bp) was detected only in the p53-IP complex of the p53-restored H1299 cells under the genotoxic stress. Statistical analysis was done with the Student's t test. **p < 0.002; ***p < 0.0001.