Journal: Nature immunology

Article Title: Activated β-Catenin in Foxp3 + Regulatory T cells Links Inflammatory Environments to Autoimmunity

doi: 10.1038/s41590-018-0236-6

Figure Lengend Snippet: (a) Representative histogram of CFSE dilution for T reg suppression assay. Yellow; Foxp3 Cre Teff only, Blue; Foxp3 Cre T reg cells and Foxp3 Cre Teff at 1:1 ratio, Red; Ctnnb1 ΔEx3/ Foxp3 Cre T reg cells and Foxp3 Cre Teff at 1:1 ratio (left). Bar graph shows percentage of suppression (right) (n=3). P values were calculated by two-sided Student’s t -test. (b) Gene expression profile of Foxp3 Cre and Ctnnb1 ΔEx3/ Foxp3 Cre T reg cells by microarray analysis. (c) Flow cytometric analysis on peripheral lymph node T reg cells from Foxp3 Cre and Ctnnb1 ΔEx3/ Foxp3 Cre mice. Quantification of gMFI for indicated molecules was shown ( Foxp3 Cre; n=4 mice, Ctnnb1 ΔEx3/ Foxp3 Cre; n=4 or 5 mice). P values were calculated by two-sided Student’s t -test. Data were represented as mean and mean +/− SD. (d) Representative immunofluorescence images of human T reg cells with PLA signal for β-catenin-Foxo1 interaction (red) and Foxp3 staining (green). Nuclei were stained with DAPI (blue). Scale bar; 5μM. Data were representative of three experiments.

Article Snippet: Antibodies and reagents used for flow cytometric analysis are listed as follows: for human samples; anti-CD4 (RPA-T4), anti-CD25 (MA251), anti-CD127 (HIL-7R-M21), anti-CD45RO (UCHL1), anti-IFN-γ (B27), anti-β-catenin (14/Beta-Catenin), anti-TCF1 (S33–966), PE-Cy™7 Streptavidin from BD Bioscience, anti-IL-10 (JES3–9D7), anti-Tbet (4B10) from BioLegend, anti-phospho Foxo1 (S256) polyclonal, anti-phospho SGK1 (T256) polyclonal (from Bioss), anti-phospho β-catenin (Ser522) (D8E11), anti-phospho AKT (Ser473) (D9E), anti-phospho GSK3β (Ser9) (D85E12), anti-Foxo1 (C29H4) from CST, anti-IFN-γ (4S.B3), anti-Foxp3 (PCH101) from eBioscience, anti-active β-catenin (8E7) from Millipore, IFN-γ secretion assay (APC), IL-10 secretion assay (PE) from Miltenyi, for mice samples; anti-Foxp3 (FJK-16s), anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-GATA3 (TWAJ), anti-RORgt (B2D) from eBioscience, anti-Helios (22F6) from BioLegend, anti-phospho Foxo1 (S256) (E1F7T), anti-phospho Foxo3a (Ser253) (D18H8), anti-phospho Foxo1(T24)/3a(T32)/4(T28) (4G6) from CST, anti-SGK1 (Y238) (from Abcam), for both human and mice samples; Zombie Aqua™ Fixable Viability dye from BioLegend.

Techniques: Suppression Assay, Expressing, Microarray, Immunofluorescence, Staining

Journal: Nature immunology

Article Title: Activated β-Catenin in Foxp3 + Regulatory T cells Links Inflammatory Environments to Autoimmunity

doi: 10.1038/s41590-018-0236-6

Figure Lengend Snippet: (a) Flow cytometric analysis of Active β-catenin, p-SGK1 (Thr256), and p-Foxo1 (Ser256) expression in human IFN-γ-producing T reg cells. T reg cells were stimulated with anti-CD3 and anti-CD28 in the presence (NaCl) or absence (Control) of additional 40 mM NaCl for 96 h followed by 4 h PMA plus iomomycin stimulation (Active β-catenin; n=18 subjects, p-SGK1; n=13 subjects, p-Foxo1; n=10 subjects). P values were calculated by two-sided Student’s t -test. (b) mRNA expression kinetics for Wnt-β-catenin target genes ( AXIN2 and TCF7 ) from nine time points were plotted and each dots represent the average of four different experiments. * P <0.05, ** P <0.01, *** P <0.001 (two-way ANOVA with Sidak’s multiple comparisons test). Data were represented as mean +/− SEM. (c) IFNG mRNA expression in human T reg cells cultured in T H 0 or T H 1 condition in the presence (NaCl) or absence (Control) of additional 40 mM NaCl for 96 h (n=19 subjects). * P <0.05, ** P <0.01, *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (d) IFNG mRNA expression in human T reg cells stimulated in the presence (NaCl) or absence (Control) of additional 40 mM NaCl with and without Wnt/inhibitor PKF115–584 (PKF) or IWR-1 (IWR) for 96 h (n=7–10 subjects). * P <0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (e) Representative flow cytometric analysis of IFN-γ and IL-10 production in human T reg cells transduced with a non-targeted shRNA or a CTNNB1 shRNA and cultured in the normal media (Control) or media supplemented with additional 40 mM NaCl (NaCl) for 96 h. Data are representative of three experiments. (f) IFNG and IL10 mRNA expression on T reg cells, and (g) frequency of IFN-γ and IL-1 0 producing T reg cells relative to control/scramble shRNA condition were shown. T reg cells were treated as in (e) ( f ; n=9 subjects, g ; n=8 subjects). * P <0.05, ** P <0.01, *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean +/− SD.

Article Snippet: Antibodies and reagents used for flow cytometric analysis are listed as follows: for human samples; anti-CD4 (RPA-T4), anti-CD25 (MA251), anti-CD127 (HIL-7R-M21), anti-CD45RO (UCHL1), anti-IFN-γ (B27), anti-β-catenin (14/Beta-Catenin), anti-TCF1 (S33–966), PE-Cy™7 Streptavidin from BD Bioscience, anti-IL-10 (JES3–9D7), anti-Tbet (4B10) from BioLegend, anti-phospho Foxo1 (S256) polyclonal, anti-phospho SGK1 (T256) polyclonal (from Bioss), anti-phospho β-catenin (Ser522) (D8E11), anti-phospho AKT (Ser473) (D9E), anti-phospho GSK3β (Ser9) (D85E12), anti-Foxo1 (C29H4) from CST, anti-IFN-γ (4S.B3), anti-Foxp3 (PCH101) from eBioscience, anti-active β-catenin (8E7) from Millipore, IFN-γ secretion assay (APC), IL-10 secretion assay (PE) from Miltenyi, for mice samples; anti-Foxp3 (FJK-16s), anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-GATA3 (TWAJ), anti-RORgt (B2D) from eBioscience, anti-Helios (22F6) from BioLegend, anti-phospho Foxo1 (S256) (E1F7T), anti-phospho Foxo3a (Ser253) (D18H8), anti-phospho Foxo1(T24)/3a(T32)/4(T28) (4G6) from CST, anti-SGK1 (Y238) (from Abcam), for both human and mice samples; Zombie Aqua™ Fixable Viability dye from BioLegend.

Techniques: Expressing, Cell Culture, Transduction, shRNA

Journal: Nature immunology

Article Title: Activated β-Catenin in Foxp3 + Regulatory T cells Links Inflammatory Environments to Autoimmunity

doi: 10.1038/s41590-018-0236-6

Figure Lengend Snippet: (a) Flow cytometric analysis of T reg cells from the mesenteric lymph nodes of wild type mice fed a normal diet (ND) or a high-salt diet (HSD) for 3 weeks. Quantification of gMFI for β-catenin and p-Foxo1/3a/4 were shown (ND; n=4, HSD; n=4). P values were calculated by two-sided Student’s t -test. (b) Flow cytometric analysis of ABC level in ex vivo T reg s of healthy controls and MS patients (HC; n=14 subjects, MS; n=11 subjects). P value was calculated by two-sided Student’s t -test. Data were represented as mean +/− SD. (c-e) Correlation plots (c); between the percentage of IFN-γ−producing T reg cells and gMFI of Active β-catenin, (d); between IFNG and PTGER2 mRNA expression, (e); between Active β-catenin level and PTGER2 mRNA expression level in healthy subjects and MS patients. Linear regression is shown with 95% confidence interval (dotted lines). Correlation statistics by two-sided Spearman rank correlation test.

Article Snippet: Antibodies and reagents used for flow cytometric analysis are listed as follows: for human samples; anti-CD4 (RPA-T4), anti-CD25 (MA251), anti-CD127 (HIL-7R-M21), anti-CD45RO (UCHL1), anti-IFN-γ (B27), anti-β-catenin (14/Beta-Catenin), anti-TCF1 (S33–966), PE-Cy™7 Streptavidin from BD Bioscience, anti-IL-10 (JES3–9D7), anti-Tbet (4B10) from BioLegend, anti-phospho Foxo1 (S256) polyclonal, anti-phospho SGK1 (T256) polyclonal (from Bioss), anti-phospho β-catenin (Ser522) (D8E11), anti-phospho AKT (Ser473) (D9E), anti-phospho GSK3β (Ser9) (D85E12), anti-Foxo1 (C29H4) from CST, anti-IFN-γ (4S.B3), anti-Foxp3 (PCH101) from eBioscience, anti-active β-catenin (8E7) from Millipore, IFN-γ secretion assay (APC), IL-10 secretion assay (PE) from Miltenyi, for mice samples; anti-Foxp3 (FJK-16s), anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-GATA3 (TWAJ), anti-RORgt (B2D) from eBioscience, anti-Helios (22F6) from BioLegend, anti-phospho Foxo1 (S256) (E1F7T), anti-phospho Foxo3a (Ser253) (D18H8), anti-phospho Foxo1(T24)/3a(T32)/4(T28) (4G6) from CST, anti-SGK1 (Y238) (from Abcam), for both human and mice samples; Zombie Aqua™ Fixable Viability dye from BioLegend.

Techniques: Ex Vivo, Expressing

Journal: BMC Cancer

Article Title: Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

doi: 10.1186/1471-2407-14-997

Figure Lengend Snippet: Transduction of locomotor muscles and the diaphragm with AAV9-d.n.FoxO. (A) Alignment of the dominant negative (d.n.) FoxO protein sequence, which includes amino acids 141–265 of mouse FoxO3a, with the corresponding mouse FoxO1 and FoxO4 amino acid sequences. FoxO1 shares ~85% amino acid sequence identity and FoxO4 75% sequence identity with the d.n.FoxO protein (shared amino acids denoted in green), all of which share >90% sequence conservation within this region. The 6 amino acid residues involved in DNA binding of the Forkhead Domain are highlighted in red and are denoted by hash marks (#) above the aligned sequences. (B and C) The AAV9 vectors driving expression of d.n.FoxO (or empty vector), which also contain an IRES driving the expression of GFP, were injected directly into the anterior hind limb compartment of mice to transduce the TA and EDL muscles, or injected directly into the intrathoracic cavity of mice to transduce the diaphragm. (B) Representative muscle cross-sections showing AAV9 transfection efficiency in the TA, EDL and diaphragm ~26 days post-injection as visualized via direct GFP fluorescence. (C) Confirmation that the d.n.FoxO protein was also expressed in muscles transduced with AAV9-d.n.FoxO was confirmed through western blot using an antibody against DsRed, which is fused to the d.n.FoxO protein.

Article Snippet: Expression plasmids for the FoxO1 triple phosphorylation mutant (Addgene Plasmid 17547, deposited by Dr. Domenico Accili) and the FoxO3a triple phosphorylation mutant (Addgene Plasmid 10711, deposited by Dr. William Sellers ), have been described previously [ ] and were injected and electroporated into mouse TA muscles as described by us previously [ ].

Techniques: Transduction, Muscles, Dominant Negative Mutation, Sequencing, Binding Assay, Expressing, Plasmid Preparation, Injection, Transfection, Fluorescence, Western Blot

Journal: BMC Cancer

Article Title: Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

doi: 10.1186/1471-2407-14-997

Figure Lengend Snippet: FoxO1 and FoxO3a regulate Cebpb , Fos and Stat3 gene expression. (A-C) Select transcripts identified via microarray as downstream targets of FoxO in locomotor muscles during C26 cancer cachexia were validated as targets in the diaphragm via qRT-PCR analyses. Data represent mean ± SE, n = 3–5 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group. (D) FoxO1 TM or FoxO3a TM expression plasmids were injected and electroporated into TA muscles of mice and harvested 4 days later for qRT-PCR analyses. Data represent mean ± SE, n = 5 animals/group. *p < 0.05 vs empty vector (EV). (E) C2C12 myoblasts were transfected with a pRL-TK-Renilla reporter plus a WT Cebpb promoter reporter construct or a Cebpb reporter construct which is mutated at two putative Forkhead binding elements, which prevents FoxO DNA binding. Following 3 days of differentiation, myotubes were treated with IL-6 (10 ng/mL) for 3 hours and harvested for measurement of firefly/renilla luciferase activity. Data represent mean ± SE, n = at least 9 wells/group. *p < 0.05 vs control. †p < 0.05 vs. WT Cebpb promoter.

Article Snippet: Expression plasmids for the FoxO1 triple phosphorylation mutant (Addgene Plasmid 17547, deposited by Dr. Domenico Accili) and the FoxO3a triple phosphorylation mutant (Addgene Plasmid 10711, deposited by Dr. William Sellers ), have been described previously [ ] and were injected and electroporated into mouse TA muscles as described by us previously [ ].

Techniques: Gene Expression, Microarray, Muscles, Quantitative RT-PCR, Control, Expressing, Injection, Plasmid Preparation, Transfection, Construct, Binding Assay, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Albumin overload in PTECs cause downregulation of Akt activity and phosphorylation of its downstream targets Foxo1 and Foxo3 : Fig-4a-c-HKC-8 cells are incubated with endotoxin free human albumin (10 mg/ml) for 6, 16 and 24 h and probed to investigate phosphorylation of Foxo1 and Foxo3. Protein expression at 6, 16 and 24 hour levels were compared to time 0 . Independent samples t-test was used for statistical comparison between two groups. Phosphorylation of Foxo1 at Ser24 and Foxo3 at Ser 253, both representing Akt phosphorylation sites were diminished at 16 and 24 hours in association with proapoptotic BCL-2 family protein, BIM expression by Mann-Whitney U test. (n=5). * = p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HKC-8 cells were transfected with a plasmid encoding GFP tagged Foxo1 (GFP-Foxo1 was a gift from Domenico Accili, Addgene plasmid # 17551) prior to albumin overload experiments to visualize translocation of Foxo1 to nucleus .

Techniques: Activity Assay, Phospho-proteomics, Incubation, Expressing, Comparison, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Inhibition of phosphorylation by Akt and nuclear translocation of Foxo1 promotes apoptosis in association with increased BIM transcription in PTECs in response to albumin overload : Fig. 5a-HKC-8 cell transfected with plasmids possessing mutations at Akt phosphorylation sites of Foxo1 and Foxo3 were subjected to albumin overload for 24 h ( n = 5) Inhibition of Foxo1phosphorylation by Akt augmented PTEC apoptosis induced by albumin overload. Independent samples T test was used for comparison of two groups. * p < 0.05, ** p < 0.01. Figure 5b-Nuclear and cytosolic Foxo1 and Foxo3 expression in response to albumin overload was examined by western blotting. Cytosol and nuclei were isolated after 24 h of albumin overload in PTECs. Foxo1 but not Foxo3 translocated to nuclei in association with albumin induced apoptosis ( n = 6). Figure 5c-Proximal tubule epithelial cells transfected with GFP-Foxo1 displayed nuclear translocation of Foxo1 and apoptosis evidenced by nuclear condensation and rounded up cells ( n = 3). Figure 5d-We demonstrated nuclear translocation of Foxo1 in Akt1/2 lox/lox SGLT2 cre mice kidneys subjected to albumin overload. In CHIP experiments, we investigated DNA-protein interactions. Nuclear extracts of PTEC subjected to albumin overload were immunoprecipitated with Foxo1 antibody. Figure 5e-The immunoprecipitated were subjected to PCR using primers against BIM promoters( n = 3). GAPDH and Histon3 was utilized as housekeeping gene for cytosol and nuclei respectively. Independent samples T test was used for comparison of two groups.

Article Snippet: HKC-8 cells were transfected with a plasmid encoding GFP tagged Foxo1 (GFP-Foxo1 was a gift from Domenico Accili, Addgene plasmid # 17551) prior to albumin overload experiments to visualize translocation of Foxo1 to nucleus .

Techniques: Inhibition, Phospho-proteomics, Translocation Assay, Transfection, Comparison, Expressing, Western Blot, Isolation, Immunoprecipitation

Journal: Scientific Reports

Article Title: Downregulation of Akt induces proximal tubule epithelial cell apoptosis via FOXO and BIM pathway in proteinuric States

doi: 10.1038/s41598-025-21498-1

Figure Lengend Snippet: Proposed pathway to proteinuria induced PTEC apoptosis: In proteinuric states, high concentrations of albumin in the glomerular ultrafiltrate downregulates phosphorylation and activation of Akt resulting in dephosphorylation of Foxo1 by Akt. Increased activation and translocation of Foxo1 to the nucleus triggers transcription of Bcl-2 family BH3 protein BIM. Activation of BIM and Bax induces mitochondrial pore formation and translocation of cytochrome c to the cytoplasm causing apoptosis via caspase-9 in PTECs.

Article Snippet: HKC-8 cells were transfected with a plasmid encoding GFP tagged Foxo1 (GFP-Foxo1 was a gift from Domenico Accili, Addgene plasmid # 17551) prior to albumin overload experiments to visualize translocation of Foxo1 to nucleus .

Techniques: Phospho-proteomics, Activation Assay, De-Phosphorylation Assay, Translocation Assay