Coexpression of FANCD2 and FOXO3a increases cellular resistance to oxidative stress. (A) PD20 cells were infected with retroviruses carrying vector, wt-FANCD2, K561R-FANCD2, FOXO3a, or both wt-FANCD2 and FOXO3a. The transduced cells were treated with H₂O₂ at the indicated doses for 16 hours. Cell survival was determined by luminescence and is shown as the percentage of the untreated cells. Mean values and SD from 3 independent determinations are shown. Statistical significance between vector and wt-FANCD2 + FOXO3a or wt-FANCD2 samples: **P < .01; *P < .05. (B) Transduced cells described in panel A were labeled with fluorescein isothiocyanate–conjugated CM-H₂DCFDA, and levels of ROS were determined by flow cytometric analysis. (C) Expression of FOXO3a-targeted genes encoding antioxidant proteins. Transduced cells described in panel A were treated with H₂O₂ at 100μM for 12 hours. Total RNA was prepared, and gene expression was analyzed by real-time PCR. Data were presented as fold change in mRNA expression relative to glyceraldehyde-3-phosphate dehydrogenase in vector-transduced cells, which was given an arbitrary level of 1.0 for each gene. Results are mean ± SD of 3 independent experiments.