(A) Western-blot of protein extracts from p53^+/+ and p53^−/− MEFs incubated in the absence (−) or presence (+) of doxorubicin (Dox, 0.2 μg/ml) for 8 hours, using antibodies to FoxO3, p21^Cip1 (a well-known target of p53), p53, and Mek1 (loading control). (B) Western-blot of protein extracts from p53^+/+ and p53^−/− MEFs incubated with Nutlin (10 μM), a p53 activator, or Doxorubicin (Dox, 0.2 μg/ml) for 0, 4, and 8 hours, using antibodies to FoxO3, p53, and β-actin (loading control). Western-blots are representative of at least two independent experiments, conducted on independent cultures of MEFs.

Real time quantitative PCR analysis of FoxO3 (A), p21^Cip1 (B), Mdm2 (C), FoxO1 (D), FoxO4 (E), and FoxO6 (F) mRNA levels in p53^+/+ and p53^−/− thymocytes, 3 hours after γ irradiation (γ IR, 10 Gy). Mean +/− SEM of two independent experiments conducted in triplicate on samples from 3–5 mice per genotype. * p<0.05, ***p<0.001 between p53^+/+ and p53^−/− thymocytes at a given time point, two-way ANOVA with Bonferroni post-test.

(A) Location and sequence of the p53 binding sites (p53-1, p53-2, p53-3, and p53-4) in the promoter and second intron of the mouse FoxO3 gene. R: G or A; W: T or A; Y: C or T; E: exon; I: intron. Also depicted are the consensus for p53 binding sites, the p53 binding site in p21^Cip1 promoter, and the mutant of critical bases in p53-4 (p53-4m). (B) ChIP on MEFs treated with Doxorubicin (Dox, 0.2 μg/ml) for 16–20 hours, using antibodies to p53 (colored bars) or control IgG (white bars). The chromatin bound to p53 or to the control IgG was analyzed by quantitative-PCR with primers surrounding a region that did not contain p53 binding sites (−), the distal p53 binding site in FoxO3 promoter (p53-1), the p53-4 binding site in FoxO3 intron 2 (p53-4), and the p53 binding site in the p21^Cip1 promoter (p53 p21^Cip1). The fold enrichment over the IgG control is represented. Mean +/− SEM of three independent experiments. **: p<0.01, one-way ANOVA. (C) ChIP on p53^+/+ and p53^−/− MEFs in the absence or presence of Doxorubicin (Dox, 0.2 μg/ml) for 6 hours, using antibodies to p53 or control IgG. The chromatin bound to p53 or to the control IgG was analyzed by quantitative-PCR with primers surrounding a region that did not contain p53 binding sites (−), the distal p53 binding site in FoxO3 promoter (p53-1), the p53-4 binding site in FoxO3 intron 2 (p53-4) and the p53 binding site in the p21^Cip1 promoter (p53 p21^Cip1). The fold enrichment over the IgG control is represented. Mean +/− SD from triplicates of one experiment. (D) Normalized activity of luciferase reporter constructs driven by 500bp surrounding the p53 binding sites p53-1 or p53-4 in p53^+/+ (black) and p53^−/− (white) MEFs. Mean +/− SEM of four independent experiments conducted in triplicate. *: p<0.05 between p53-4 and control in p53^+/+ MEFs, **: p<0.01 between p53^+/+ and p53^−/− MEFs for p53-4, one-way ANOVA. (E) Normalized activity of luciferase reporter constructs driven by the region surrounding the p53-4 binding site or by the region surrounding the p53-4 binding site in which the p53 binding site was mutated (p53-4m) in p53^+/+ (black) and p53^−/− (white) MEFs. Mean +/− SEM of three independent experiments conducted in triplicate. **: p<0.01 between p53-4m and p53-4 in p53^+/+ MEFs, one-way ANOVA.

(A) Percent cleaved caspase 3-positive cells in E1A-transformed MEFs (FoxO3^+/+p53^+/+ (+/+), FoxO3^−/−, and p53^−/−) in the absence of treatment (−) or in response to Nutlin (N), serum starvation (S), and Nutlin + serum starvation (N+S). Mean +/− SEM of three independent experiments. (B) Real time quantitative PCR analysis of Bim mRNA levels in p53^+/+ and p53^−/− thymocytes, 3 hours after γ irradiation (γ IR, 10 Gy). Mean +/− SEM of two independent experiments conducted in triplicate on samples from 3–5 mice per genotype. * p<0.05 between p53^+/+ and p53^−/− thymocytes at a given time point, two-way ANOVA with Bonferroni post-test.

Real time quantitative PCR analysis of FoxO3 (A), p21^Cip1 (B), Mdm2 (C), FoxO1 (D), FoxO4 (E), and FoxO6 (F) mRNA levels in p53^+/+ and p53^−/− MEFs in response to 4 and 8 hours of treatment with Nutlin (10 μM) or Doxorubicin (Dox, 0.2 μg/ml). Mean +/− SEM of two independent experiments conducted in triplicate. * p<0.05, ** p<0.01, ***p<0.001 between p53^+/+ and p53^−/− MEFs at a given time point, two-way ANOVA with Bonferroni post-test.

(A) Schematic of the p53 knock-in alleles used. p53^LSL-WT: inducible allele encoding a form of wildtype p53. p53^LSL-25,26: inducible allele encoding a transcriptionally-impaired mutant of p53 in which leucine 25 is replaced by a glutamine and tryptophan 26 is replaced by a serine. p53^LSL-VP16: inducible allele encoding a mutant of p53 in which the transactivation domains (AD1 and AD2) are replaced by the transactivation domain of VP16. AD: activation domain; Olig.: oligomerization domain. The star indicates the location of the 25,26 mutation. (B) Northern-blot analysis of MEFs in which the endogenous allele of p53 has been replaced by an allele encoding inducible forms of WT p53 (p53^LSL-WT), a transcription-deficient mutant (p53^LSL-25,26), or a mutant of p53 in which the transactivation domains of p53 were replaced by that of VP16 (p53^LSL-VP16). The addition of an adenovirus containing Cre recombinase (Ad-Cre) allows the deletion of the Lox-STOP-Lox (LSL) cassette upstream of each allele and allows the expression of each p53 variant. Cells were exposed to 8 hours of doxorubicin (Dox, 0.2 μg/ml). Northern-blots were analyzed with a probe to FoxO3, p21^Cip1 (a known target of p53), and GAPDH (loading control).

(A) Percent BrdU-positive cells in FoxO3^+/+p53^+/+ (+/+), FoxO3^−/− and p53^−/− MEFs in the presence or absence of doxorubicin (Dox, 0.2 μg/ml) for 24 hours. Mean +/− SEM of three independent experiments, two of which were conducted with independent MEF cultures from distinct animals. ***p<0.001 between +/+ and p53^−/− MEFs for the same treatment; ns: non significant between +/+ and FoxO3^−/− MEFs for the same treatment, two-way ANOVA with Bonferroni post-test. (B) Percent BrdU-positive cells in FoxO3^+/+p53^+/+ (+/+), FoxO3^−/− and p53^−/− MEFs in the presence or absence of Nutlin (10 μM) for 24-36 hours. Mean +/− SEM of three independent experiments. *p<0.05, ***p<0.001 between +/+ and p53^−/− MEFs for the same treatment; ns: non significant between +/+ and FoxO3^−/− MEFs for the same treatment, two-way ANOVA with Bonferroni post-test. (C) Percent BrdU-positive cells in FoxO3^+/+p53^+/+ (+/+), FoxO3^−/− and p53^−/− MEFs in the presence or absence of chronic treatment of H₂O₂ or hydroxyurea (HU). Mean +/− SEM of two independent experiments with different lines of MEFs. **p<0.01, ***p<0.001 between +/+ and FoxO3^−/− or p53^−/− MEFs for the same treatment; ns: non significant between +/+ and FoxO3^−/− MEFs for the same treatment, two-way ANOVA with Bonferroni post-test. (D) Real time quantitative PCR analysis of p27^Kip1 mRNA levels in p53^+/+ and p53^−/− MEFs in response to 4 and 8 hours of treatment with Nutlin (10 μM) or Doxorubicin (Dox, 0.2 μg/ml). Mean +/− SEM of two independent experiments conducted in triplicate. ***p<0.001 between p53^+/+ and p53^−/− MEFs at a given time point, two-way ANOVA with Bonferroni post-test. (E) Percent BrdU-positive cells in MEFs infected with control lentiviruses (PSR) or with lentiviruses expressing an shRNA to FoxO family members (PSR “pan FoxO”) in the presence or absence of Nutlin (Nutlin, 10 μM) for 24 hours. The data are expressed as fold decrease, respective to the value obtained in the absence of Nutlin. Mean +/− SEM of two independent experiments. ** p<0.01, between PSR and PSR “pan” FoxO-infected MEFs in the presence of Nutlin, two-way ANOVA with Bonferroni post-test. (F) Western-blot of protein extracts from MEFs infected with control lentiviruses (PSR) or with lentiviruses expressing an shRNA to FoxO family members (PSR “pan FoxO”), using antibodies to FoxO1, FoxO3, FoxO4, FoxO6, and β actin.

(A). Percent survival of mice with different alleles of FoxO3 in the p53^−/− background as a function of time. Kaplan-Meier survival curves with the number of mice indicated for each genotype. p = 0.10, logrank test. (B) Percent survival of mice with different alleles of FoxO3 in the p53^+/− background as a function of time. Kaplan-Meier survival curves with the number of mice indicated for each genotype. p = 0.13, logrank test. (C) Tumor types and glomerulonephritis in mice with different alleles of FoxO3 in the p53^+/− and p53^−/− background. The number of mice is indicated for each genotype. (D) Examples of sarcomas and carcinomas in compound FoxO3/p53 mutant mice. Main panels: 50x. Insets: 630x. i: subcutaneous fibrosarcoma in a FoxO3^+/−p53^−/− mouse; ii: osteosarcoma in the leg of a FoxO3^−/−p53^−/− mouse; iii: colon carcinoma in a FoxO3^+/−p53^−/− mouse; iv: uterine carcinoma in a FoxO3^+/−p53^+/− mouse; v: breast carcinoma in a FoxO3^+/−p53^+/− mouse; vi: muscle carcinoma in the arm of a FoxO3^+/−p53^+/− mouse.