Fig 2: FoxO4 inhibits CDX2 expression by directly targeting the CDX2 promoter region. (A) Serially truncated CDX2 promoter constructs were cloned into pGL3‐luciferase reporter plasmids and transfected into GES‐1 cells. Four hours after transfection, the cells were treated with CDCA (200 μM) for 24 hr, and the relative luciferase activities were determined 72 hr later. (B) A ChIP assay demonstrated the direct binding of FoxO4 to the CDX2 promoter in GES‐1 cells. M: Marker. (C,D) qRT‐PCR of the ChIP products validated the binding capacity of FoxO4 to the CDX2 promoter. Means ± SEM of a representative experiment (n = 3) performed in triplicate is shown. *p < .05; **p < .01; ***p < .001; ns, not significant

Fig 3: FOXO3 regulates PERK expression in MCF-7 and MEFs. a Expression levels of protein and mRNA in WT and Foxo1/3/4−/− MEFs in response to epirubicin. Western blotting was performed to determine the protein expression levels for P-Foxo3 (T32) (95 kDa), Foxo3 (85 kDa), P-Foxo1 (T24) (79 kDa), Foxo1 (79 kDa), Foxo4 (54 kDa), P-Perk (T981) (140 kDa), Perk (140 kDa), P-eIF2a (S51) (38 kDa), eIF2a (38 kDa) and β-Tubulin (55 kDa). Foxo3, Foxo1, Foxo4 and Perk mRNA levels were investigated by RT-qPCR. Three technical repeats were conducted in one experiment, and the data were normalised to L19 and displayed as means ± SEM. (n = 6; two-tailed t-test). Representative RNA expression profiles of at least three independent experiments are shown (right panel). b Expression levels of protein and mRNA in MCF-7 cells overexpressing FOXO3. P-FOXO3 (T32) (95 kDa), FOXO3 (85 kDa), P-FOXO1 (T24) (79 kDa), FOXO1 (79 kDa), P-PERK (T981) (140 kDa), PERK (140 kDa), P-eIF2a (S51) (38 kDa), eIF2a (38 kDa) and β-Tubulin (55 kDa) as loading control. FOXO3, FOXO1, FOXO4 and PERK mRNA were determined by RT-qPCR. Three technical repeats were conducted in one experiment, and the data were normalised to L19 and displayed as means ± SEM. (n = 6; two-tailed t-test). (right panel). c FOXO3-binding site on human PERK promoter. ChIP-sequencing data of FOXO3-binding in DLD1 colon carcinoma cells [26] were used for predicting FOXO3-binding sites using the Integrative Genomics Viewer (Version 2.3.88) and the hg19 UCSC Genome Browser 45. The predicted binding profiles of FOXO3, and the locations of the designed ChIP primer pairs are aligned to the human PERK promoter. MCF-7 cells treated with 1 μM epirubicin for 0, 4, 8 and 24 h were used for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXO3 antibody. After reversal of cross-linking, the co-immunoprecipitated DNA was amplified by qRT–PCR, using primers amplifying the FOXO3-binding-site-containing region. Data are displayed as means ± SEM (n = 3; one-way ANOVA). d ChIP analysis of FOXO3 binding on PERK promoter in MCF-7 and MCF-7-EpiR cells transfected with empty vector or FOXO3 expression vector. Representative RNA expression profiles of at least three independent experiments are shown. Three technical repeats were conducted in one experiment, and data were normalised to IgG and displayed as means ± SEM (n = 6; two-tailed t-test). Significant: *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: Changes of ß-catenin and SIRT1 protein in spinal cord injury rats. (a, b) The (a) ß-catenin and (b) SIRT1 expression in the spinal cord tissue of SCI rat at different time points were detected by immunofluorescence; (c) Western blot were used to determine the expression of ß-catenin, SIRT1, FOXO4, and Caspase-3 in SCI rats. N = 5 per group. *P < 0.05; **P < 0.01; ***P < 0.001vs. sham group. SCI: spinal cord injury.

Fig 5: FOXO1 is a Specific Regulator of HIV Latency Establishment.a, Schematic representation of HIVGKO dual-labeled HIV-1 reporter. K562(Cas9) cells were treated with increasing concentrations of the FOXO1 inhibitor AS1842856 just after HIVGKO infection. After 3–4 days, the percentage of latent and productively infected cells were quantified by FACS. In upper panel, amount of productively or latently infected cells (bars) and total cell viability (blue dotted line) of a representative experiment. In the lower panel, ratios of productive versus latent populations relative to the total infection rate upon increasing concentrations of AS1842856 treatments. Data are represented as mean ± SD of triplicate values, representative of n=3 independent experiments. b, FOXO1, FOXO3 and FOXO4 CETSA-melting curve shifts upon the presence or absence of AS1842856 1,000 nM in K562(Cas9) cells. Band intensities obtained from western blot analysis were normalized to the highest western blot signal. Relative FOXO-band intensities were plotted against corresponding incubation temperatures and a nonlinear least-squares regression fit was applied. Data represent the mean ± SD of n=3 independent experiments, except for FOXO4 (n=2). c, Efficiency of FOXO1, FOXO3 and FOXO4 knockdowns with two different sgRNAs determined by western blot. Cells transduced with NC (negative control) sgRNA lentiviruses were used as a control. Representative of n=3 independent experiments, except for FOXO3 (n=2). d, In the left graph, percentage of productive or latent cells relative to the total infection rate and cell viability in the different single knockdown K562(Cas9) cell lines. In the right graph, ratios of productive versus latent populations in K562(Cas9) cell lines with knockdown of FOXO1, FOXO3, or FOXO4. Data are represented as mean ± SD of triplicate values, representative of n=3 independent experiments. e, Ratios of productive versus latent populations upon increasing concentrations of AS1842856 in the WT K562(Cas9) or in the FOXO1-knockdown cell lines. Data are represented as mean of n=2 independent experiments fitted into a model.