Oxidative stress–induced formation of the FANCD2-FOXO3a complex. (A) FOXO3a colocalizes with FANCD2 after H₂O₂ treatment in normal (JY) lymphoblasts. Cells were treated with H₂O₂ (0.5mM) for 6 hours and stained with antibodies against FOXO3a and FANCD2. Colocalization of nuclear FOXO3a with FANCD2 is shown as merged images. (B) Whole-cell lysates of normal lymphoblasts (JY) and HeLa cells treated with 0.5mM H₂O₂ for 6 hours were subjected to immunoprecipitation (IP) with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by immunoblotting (IB) analysis with antibodies against FOXO3a, FOXO1, FOXO4, and FANCD2. (C) HeLa cells were transduced with vector or Flag-FOXO3a and treated with 0.5mM H₂O₂ for 6 hours. Cell lysates were prepared, immunoprecipitated using anti-Flag agarose, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotted. Blots were probed with antibodies against FANCD2 or Flag. (D) Normal human lymphoblasts (JY) were treated with H₂O₂ (0.5mM for 6 hours), IR (5 Gy), or MMC (0.5μM for 18 hours), and whole-cell lysates were subjected to IP with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by IB analysis with antibodies against FOXO3a and FANCD2. (E) HSC72 (human FA-A lymphoblast) cells were transduced with retrovirus carrying empty vector (MIEG3) or FANCA, and treated with H₂O₂ at 0.5mM for 6 hours. Cell extracts were subjected to IP with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by IB analysis with antibodies against FOXO3a and FANCD2. (F) PD20 (human FA-D2 lymphoblast) cells transduced with retrovirus carrying empty vector wt-FANCD2 or K561R-FANCD2 were treated with H₂O₂ at 0.5mM for 6 hours. Cell extracts were prepared, immunoprecipitated using anti-Flag antibody conjugated to agarose, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotted with antibodies against FANCD2 and FOXO3a.