Figure 4.

H₂O₂ induces nuclear translocation and activation of FOXO4. (A) DLD1 cells, transfected with HA-FOXO4, were maintained in the presence of serum and left untreated (left panel) or treated with 100 μM of H₂O₂ for 60 min (middle and right panels). Cells were fixed and HA-FOXO4 was stained. The middle panel shows differential response to oxidative stress with respect to localization. Same results were obtained using 200 or 400 μM of H₂O₂. (B) DLD1 cells, transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4, were treated with indicated concentrations of H₂O₂ for 16 h and subjected to luciferase assays. Equal expression was tested by Western blot. Data represent the average of three independent experiments. (C) DLD1 cells were transfected with 6xDBE-luciferase together with the indicated constructs, and subjected to luciferase assays as described in panel B. (D) DLD-1 cells were transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4 and absence or presence of dominant-negative Ral (RalN28), and subjected to luciferase assays as described in panel B. (E) A14 cells were transfected with 6xDBE-luciferase in the presence of HA-FOXO4 or the indicated mutant constructs and either in the presence or absence of dominant-negative Ral (RalN28), and subjected to luciferase assays as described in panel B. (F) JNK1,2−/− MEFs and wt MEFs were transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4, HA-JNK1 or both, and subjected to luciferase assays as described in panel B. (G) JNK1,2−/− MEFs, transfected with pSODLUC-3340 with or without JNK3, were treated with indicated concentrations of H₂O₂ for 16 h, and subjected to luciferase assays as described in panel B. (H) wt MEFs and JNK1,2−/− were transfected with 6xDBE-luciferase together with indicated amounts of HA-RlfCAAX, and subjected to luciferase assays as described in panel B. (I) A14 cells were transfected with the indicated constructs and a puromycin selection vector. At 36 h after transfection, cells were put on 2 μg/ml puromycin and either cultured in medium containing FCS with glucose or medium with FCS lacking glucose. After 48 h of glucose deprivation, cells were harvested, stained with rhodamine-1,2,3 and analyzed for mitochondrial membrane stability.