Fig. 6. FOXO3A requires GSK-3 activity for full transcriptional activity.

a 293T HEK cells were transfected with a reporter plasmid with Luciferase driven by a wild type (wt) or FOXO3A binding site mutant (FoxoBSmut) Puma promoter fragment, a Renilla reporter plasmid as internal control and a construct encoding FOXO3A-TM or empty vector. Eight hours after transfection, the cells were treated with CT98014 (CT, 0.75 µM,) or DMSO for 18 h. Luciferase activity was analyzed and normalized to Renilla activity. Error bars represent SD from technical replicates. b Ba/F3 cells expressing CRISPR/Cas9 constructs targeting Luciferase or a Foxo3a^−/− single-cell clone were infected with retrovirus expressing human FOXO3A-TM or none (empty vector, EV). The cells were deprived of IL-3+/− CT98014 (CT, 0.75 µM) for 18 h, then stained with Annexin-V-FITC and analyzed by flow cytometry. Error bars represent SD from technical replicates. This experiment was done together with the one shown in Fig. 5b, and the controls are identical. c HEK 293T cell were transfected with constructs encoding wild-type, FLAG-tagged FOXO3A (wt) or FLAG-tagged FOXO3A-TM (TM) or control vector (−). Eight hours later, the cells were treated with CT98014 (0.75 µM) or left untreated for 18 h. The cells were lysed and 2% of the lysate were kept as input. FLAG immunoprecipitation was performed with the remaining lysate. After washing and elution with 3xFLAG peptide, eluate and input fractions were analyzed by western blotting with the antibodies indicated.