1. FOXO3a Is Activated in Response to Hypoxic Stress and Inhibits HIF1-Induced Apoptosis via Regulation of CITED2 
Walbert J. Bakker, Isaac S. Harris, Tak W. Mak 
Molecular Cell 
Volume 28, Issue 6, Pages (December 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Hypoxia Induces Foxo3 Expression in an Hif1-Dependent Manner
(A and B) MEF (A) and NIH 3T3 (B) cells were cultured under hypoxic conditions for 24 or 48 hr, respectively. Cell lysates were prepared at the indicated time points. Membranes were immunostained to detect total and phospho-S253 Foxo3. β-tubulin was used as a loading control.
(C and D) Cytoplasmic mRNA was harvested from MEF (C) and NIH 3T3 (D) cells cultured for 8 or 24 hr under hypoxia, respectively. Foxo3 transcript levels were determined by qPCR. Data shown are the fold induction of mRNA under hypoxia compared to normoxia. Error bar values are the mean ± SD of at least three independent experiments.
(E) Vegf, Foxo1, Foxo3, and Foxo4 mRNA levels in WT and Hif1α−/− MEFs were measured by qPCR at the indicated time points during incubation under hypoxia.
Error bar values are the mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Foxo3 Is Activated and Stimulates Transcription of Cited2 during Hypoxia
(A) Status of Foxo3 in cytoplasmic (C.E.) and nuclear (N.E.) extracts of MEFs treated with 15 μM LY (LY) or incubated for 8 hr in hypoxia. Total Foxo3 Ab detects both the phosphorylated fraction (upper band, top panel) and the nonphosphorylated fraction (lower band, top panel). β-tubulin (bottom panel) was used as a loading control for C.E. The indicated background bands in the top panel served as loading controls for N.E. Immunostaining with anti-S253-Foxo3 Ab is shown in the middle panel.
(B) DBE reporter assay. NIH 3T3 cells were transfected with a reporter plasmid containing a 6× DBE repeat and after 24 hr were cultured under normoxia or hypoxia for 48 hr. Results are expressed as fold induction compared to basic promoter activity under normoxic conditions.
(C) DBE reporter assay as in (B) but now using 293T cells and overexpression of FOXO3a(A3).
(D) DBE reporter assay performed in WT and Hif1α−/− MEFs. The level of DBE promoter activity is similar in WT and Hif1α−/− MEFs maintained under normoxic conditions. Results are expressed as fold induction compared to basic promoter activity under normoxic conditions. Data shown represent the mean of duplicates in two independent experiments.
(E) MEFs were retrovirally transduced with HA-FOXO3a(A3):ER or control vector (pBabe). Stable clones were treated for 2 or 6 hr with 100 nM 4OHT. Cited2 mRNA levels were analyzed by qPCR. Fold increase represents 4OHT-treated compared to nontreated cells.
(F) Schematic representation of the Cited2 promoter. Two DBEs, two Hif1-responsive elements (HRE), and one Stat5-responsive element (SRE) were identified. Alignment (data not shown) revealed that all sites, except the −2043 DBE, were conserved between humans and mice.
(G) ChIP assay. MEFs stably expressing HA-FOXO3a(A3):ER were treated with 100 nM 4OHT for 6 hr. FOXO3a(A3) complexes were precipitated using anti-HA Ab. Anti-Myc Ab was used as a negative control. Promoter fragments were detected by PCR.
(H) Human CITED2 reporter assay. NIH 3T3 cells overexpressing FOXO3a(A3) were cultured for 48 hr under normoxia or hypoxia as indicated. CITED2 promoter activity presents fold increase compared to promoter activity under normoxic conditions.
(I) CITED2 reporter assay performed in WT and Hif1α−/− MEFs, as described in (D).
(J) Mouse Cited2 reporter assay as in (H), except that the WT and −872 DBE mutated mouse Cited2 promoters were examined.
Error bar values are the mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 FOXO3a(A3) Inhibits Hif1 during Hypoxia
(A) Stable FOXO3(A3):ER or control MEFs were treated for 8 hr with 100 nM 4OHT under normoxia or hypoxia. Cytoplasmic RNA was isolated, and the expression of the indicated FOXO3a(A3) target genes was measured by qPCR.
(B) Expression of Hif1 targets was analyzed as in (A). For (A) and (B), results are expressed as the fold change compared to normoxia.
Error bar values are the mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Suppression of Foxo3 mRNA Activates Hif1
(A and B) NIH 3T3 cells were transfected with a control or Foxo3 siRNA. Knockdown of Foxo3 was verified by western blot (A) and qPCR (B).
(C and D) NIH 3T3 cells were transfected with a Foxo3 siRNA and after 24 hr were cultured under hypoxia for 48 hr. Transcript levels of Foxo3, Cited2, and Vegf (C), as well as Nix, Rtp801, and Bnip3 (D), were determined by qPCR.
(E) Levels of Nix, Rtp801, and Bnip3 mRNAs were determined by qPCR in WT and Hif1α−/− MEFs cultured for the indicated times under hypoxia.
(F) Cell death was assayed in WT and Hif1α−/− MEFs cultured for 16 hr under hypoxia. Values shown are the percentage of Annexin V/PI double-positive cells as determined by flow cytometry. ∗p <
Error bar values are the mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5 Foxo3 and Cited2 Stimulate Cell Survival under Hypoxia
(A) MEFs were transfected with control or Foxo3 siRNA and after 24 hr were cultured for 24 hr under hypoxia. Cell death was determined by Annexin V/PI staining as for Figure 4F. ∗p <
(B) Assays as in (A), except that NIH 3T3 cells were cultured for 48 hr under hypoxia. ∗p <
(C) Assays as in (A), except that cells were transfected with an siRNA for Cited2. ∗p <
(D) Validation of Cited2 siRNA. NIH 3T3 cells were transfected with control or Cited2 siRNA and after 24 hr were cultured under hypoxia for 24 hr. Cited2 mRNA levels were determined by qPCR.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
FOXO3a Inhibits HIF1-Induced Apoptosis in MCF7 Breast Cancer Cells
(A) Two different HIF1α siRNAs were tested for their efficiency to knock down endogenous HIF1α protein in MCF7 cells. siRNA-transfected cells were incubated under hypoxia for 24 hr, 24 hr after transfection. Protein expression was analyzed by western blot. β-tubulin serves as a loading control.
(B) The effect of HIF1α suppression on target gene expression analyzed by qPCR. Conditions are similar to those in (A). mRNA expression of the HIF1 targets VEGF, RTP801, and NIX was tested.
(C) Cell death in the cultures in (B) was determined by flow cytometry after 36 hr culture under hypoxia. Values shown are the percentage of Annexin V/PI double-positive cells as determined by flow cytometry. ∗p <
(D) 293T cells were transfected with an HA-FOXO3a or control plasmid in the presence or absence of a FOXO3a siRNA. Suppression of FOXO3a protein expression was validated by western blot.
(E) MCF7 cells were transfected with a FOXO3a siRNA and after 48 hr were cultured under hypoxia for 16 hr. Efficiency of FOXO3a knockdown and its effect on CITED2, VEGF, NIX, and RTP801 expression was determined by qPCR.
(F) Western blot analysis of protein expression after FOXO3a knockdown under hypoxia. MCF7 cells were transfected with a control or FOXO3a siRNA, and after 24 hr were treated with hypoxia for 24 hr. β-tubulin was used as a loading control.
(G) Cell death in the cultures in (E) was determined as described in (C). ∗p <
(H) Validation of siRNAs for RTP801 and NIX by western blot in MCF7 cells cultured for 24 hr under hypoxia (24 hr after transfection). NIX siRNA served as a negative control for RTP801 siRNA and vice versa.
(I) Cell death was determined as described in (C) in the cultures of (H). Data shown are the percentage of Annexin V/PI double-positive cells and represent the mean of duplicates in two independent experiments.
Error bar values are the mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 FOXO3a Controls HIF1-Induced Apoptosis during Hypoxic Stress
Hypoxia activates HIF1 through stabilization of the HIFα subunit. The PTEN/PI3K pathway also regulates HIF1 activity through stimulation of HIF1α translation (via mTOR), and via inhibition of the Forkhead transcription factor FOXO3a (this study). FOXO3a is part of a negative feedback loop that controls HIF1 activity, as FOXO3a mRNA levels increase during hypoxia in an HIF1-dependent way, and because FOXO3a stimulates transcription of CITED2, a negative regulator of HIF1 activity. In response to hypoxic stress, FOXO3a inhibits HIF1-induced apoptosis in normal and breast cancer cells by reducing the expression of the proapoptotic HIF1 targets NIX and RTP801.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions