Down-regulation of antioxidant genes in FA BM cells. (A) Hypersensitivity of FA BM progenitors to H₂O₂. BM cells from 5 FA-A and 2 FA-C patients, as well as 5 healthy donors, were treated with 100μM H₂O₂ for 2 hours followed by a colony-forming assay. Colony numbers were counted 10 days after plating. Results are means ± SD of 3 independent experiments. (B) Hypersensitivity of FA LCLs to H₂O₂. FA-A LCLs and cells corrected with the FANCA gene were treated with increasing doses of H₂O₂ for 2 hours followed by culture in fresh medium for an additional 24 hours. Cells were then analyzed for cell survival. (C) BM cells from 5 FA-A, 1 FA-B, 2 FA-C, 1 FA-D1, 1 FA-I, and 1 FA-J patients and healthy donors were used for pathway-specific (oxidative stress and antioxidant defense) array analysis. Eighty-four genes involved in antioxidative stress response were analyzed. The data represent the fold down-regulation of the indicated genes in FA samples relative to healthy donors. Fold down-regulation = −1/(fold difference), where fold difference = [2^−ΔΔCt (FA)]/[2^−ΔΔCt (healthy)]. (D) RT-PCR analysis of antioxidant gene expression in FA-A BM cells. RNA was extracted from BM cells from 2 FA-A patients and 2 healthy donors followed by RT-PCR analysis. (E) Western analysis of antioxidant gene products in FA-A BM cells. Protein lysates were prepared from cells described in panel B followed by Western blot analysis using the indicated Abs. Relative mRNA and protein levels were quantified using ImageJ software (NIH). The results were plotted after normalization with β-actin as an internal control.