Fig. 3.

Nuclear FOXO3a represses transcriptional activity. (A) Pten^−/− and Pten^+/− cells were cotransfected with the luciferase reporter construct containing a 6× repeat of the Daf16-binding elements (6×DBE-Luciferase) and the normalization construct (pTK-rLuc). At 24 h after transfection, cells were harvested and data were reported as the ratio of firefly/Renilla luciferase activity. (B) Pten^−/− cells were cotransfected with the HRE-Luciferase reporter gene construct and the normalization construct (pTK-rLuc). At 24 h after transfection, cells were infected with null adenovirus or adenovirus expressing WT FOXO3a or the constitutively active FOXO3a (AAA) (100 pfu/μl) and subsequently exposed to 21% O₂ or 1.5% O₂ for 16 h. Firefly luciferase activity was normalized to Renilla luciferase activity and data reported as fold induction over 21% O₂ AdNull. (C) Pten^−/− cells were transfected with a GAL4 (amino acids 1–147) DNA-binding domain fused to HIF-1α (amino acids 531–826) construct and a reporter gene construct encoding five GAL4-binding sites. After transfection, cells were infected with adenoviruses. Relative luciferase expression is the ratio of luciferase/total protein levels normalized to AdNull infected cells. (D) Pten^−/− cells were cotransfected with the HRE-Luciferase reporter gene construct and the normalization construct (pTK-rLuc) and subsequently exposed to 21% O₂ or 1.5% O₂ for 16 h ± 20 nM LMB. Firefly luciferase activity was normalized to Renilla luciferase activity, and data are reported as fold induction over untreated (−LMB) normoxic cells.