(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 p27Kip1 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.