Figure 5.

TNFα induces FOXO4 transcriptional activity. (A) A14 cells were transfected with the HA-FOXO4 with or without HA-RalN28, together with 6xDBE-luciferase and Tk-renilla. Cells were treated with increasing concentrations of TNFα (5, 10 and 20 ng/ml). Pretreatment with NAC was performed by adding 10 mM of NAC 16 h before treatment with TNFα. At 16 h after TNFα treatment, luciferase activity was measured. Data represent the average of three independent experiments. (B) JNK1,2−/− MEFs were transfected with HA-FOXO4 with or without HA-JNK1 together with 6xDBE-luciferase and Tk-renilla, and treated for 16 h with TNFα (20 ng/ml). Luciferase activity was measured as described in panel B. (C) A14 cells were treated with 10 mM NAC and the next day treated with TNFα (20 ng/ml) or H₂O₂ (100 μM) for the indicated time points. JNK activity was measured by a GST-Jun pull-down. (D) A14 cells were treated with TNFα (20 ng/ml), H₂O₂ (100 μM) or insulin (1 μg/ml) for the indicated time points. JNK activity was measured by means of a GST-Jun pull-down and PKB activity was monitored using the S473 phosphospecific antibody.