Down-regulation of antioxidant genes is associated with a selective increase in promoter DNA damage in FA cells. (A) Increased promoter DNA damage in antioxidant genes in FA-A cells. FA-A cells transduced with empty vector or cDNA-encoding FANCA, as well as a normal control, were treated with 100μM H₂O₂ for 2 hours followed by 12 hours of culture in fresh medium. Genomic DNA was isolated followed by FPG cleavage and qPCR using primers specific for the promoters of the indicated genes. The percentage of intact DNA represents the ratio of PCR products after Fpg cleavage to those present in uncleaved DNA. (B) DNA damage in the coding sequences of antioxidant defense genes. The same analysis was applied as described in panel A using primers specific for exons of the indicated genes. (C-D) Increased 8-oxodG accumulation in the promoters of antioxidant genes in FA cells. FA-A or gene-corrected cells were treated with increasing doses of H₂O₂ for 2 hours and released into fresh medium for another 12 hours, followed by ChIP using an Ab against 8-oxodG. Precipitated samples were then subjected to PCR using primers specific for promoter regions of (C) GPX1 or (D) TXNRD1 gene. Representative images (left) and quantifications (right) are shown. The intensities of DNA bands were quantified using ImageJ software (NIH). Results are means ± SD of 3 independent experiments.