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: Experimental and Therapeutic Medicine

Article Title: Enhanced SLC2A4 expression mitigates insulin resistance in gestational diabetes mellitus via the FoxO signaling pathway in vitro

doi: 10.3892/etm.2025.12956

Figure Lengend Snippet: Role of the FoxO signaling pathway in pc-SLC2A4-induced changes in HG-induced HTR-8/SVneo cells. (A) Screen of targeted pathways related to gestational diabetes mellitus and SLC2A4 (five common genes identified through the CTD/GeneCards/DisGeNet database) using Kyoto Encyclopedia of Genes and Genomes, suggesting a critical role for FoxO signaling pathway. (B) The expression levels of p-FoxO1, FoxO1, p-FoxO3a and FoxO3a were analyzed by western blotting, GAPDH was used as the internal control for the results. The expression of INS and INSR was analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting. (E) Glucose uptake was detected by glucose uptake assay. (F) Cell viability was measured using a Cell Counting Kit-8 assay. *** P<0.001 vs. Control; ns P>0.05 vs. HG; ### P<0.001 vs. HG + pc-NC; $ P<0.05, $$ P<0.01 and $$$ P<0.001 vs. HG + pc-SLC2A4. SLC2A4, solute carrier family 2 member 4; HG, high glucose; ns, not significant; NC, negative control; OD, optical density; INS, insulin; INSR, INS receptor; p, phosphorylated; FoxO, Forkhead box O.

Article Snippet: The membranes were then incubated overnight at 4 ̊C with primary antibodies against SLC2A4 (cat. no. 2213S; Cell Signaling Technology, Inc.), INSR (cat. no. 3025S; Cell Signaling Technology, Inc.), INS (cat. no. ab181547; Abcam), phosphorylated (p)-FoxO1 (cat. no. 9461S; Cell Signaling Technology, Inc.), FoxO1 (cat. no. 2880S; Cell Signaling Technology, Inc.), p-FoxO3a (cat. no. 13129S; Cell Signaling Technology, Inc.), FoxO3a (cat. no. 12829S; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 97166; Cell Signaling Technology, Inc.) (all 1:1,000 dilution).

Techniques: Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Negative Control

Journal: EBioMedicine

Article Title: miR-378 Activates the Pyruvate-PEP Futile Cycle and Enhances Lipolysis to Ameliorate Obesity in Mice

doi: 10.1016/j.ebiom.2016.01.035

Figure Lengend Snippet: miR-378 activates pyruvate-PEP futile cycle via the Akt1-FoxO1-PEPCK pathway. (A) Phosphorylated (p-) and total (t-) level of Akt1 and FoxO1 in skeletal muscle of Wt and Tg mice determined by Western blot. GAPDH served as loading control. (B) Adenovirus containing constitutively active Akt1 (Myc-tagged Myr-Akt1) was injected into the tail-vein of Wt and Tg mice. Scramble adenovirus served as control (Con). Ten days after infection, antibodies detected expression of Myc-tagged Myr-Akt1 and phosphorylated (p-) and total (t-) levels of FoxO1 in skeletal muscle by Western blot. β-actin served as loading control. (C) Relative expression of the Pepck gene in gastrocnemius muscle of Wt and Tg mice described in (B). (D–F) Expression of lipolytic genes in WAT, BAT, and skeletal muscle of mice described in (B). (G) Free-fatty acids (FFA) in serum of mice described in (B). Data are presented as means ± s.e.m. from 5 male mice in each group. The statistical significance of the difference between two means was calculated using Student's unpaired t -test. * P < 0.05. NS, statistically non-significant. See also Fig. S6.

Article Snippet: Protein lysates were resolved by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and immunoblotted with primary antibodies against total Akt1 (Cell Signaling 2938, 1:1000), phosphorylated Akt1 (Santa Cruz sc7985, 1:2000), total FoxO1 (Cell Signaling 2880, 1:300), phosphorylated FoxO1 (Cell Signaling 9464s, 1:300), Myc-tag (Millipore 05–724, 1:2000), GAPDH (Millipore, 1:10,000), or β-actin (Sigma, 1:20,000).

Techniques: Western Blot, Control, Injection, Infection, Expressing

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: Generation of FoxO1-S253A/A mice and postprandial hyperglycemia. (A) The genomic locus of FoxO1 in mouse genome. (B) Gene-targeting strategy for generation of FoxO1-S253A mutation via embryonic stem (ES) cell and homologous recombination (HR). (C and D) Southern blot was performed for screening the 5′- and 3′-end HR in FoxO1 genomic loci. DNA sequencing of endogenous FoxO1 loci of negative and positive ES cells. (E) Body weight curve of mice of control wild-type (WT), heterozygous (A/+), and homozygous (A/A) mice at the ages of 4 to 16 weeks. (F) Effect of insulin on FoxO1 protein and phosphorylation (p) in hepatocytes. Primary hepatocytes were isolated from the control and A/A mice and treated with 100 nM insulin for 30 minutes, and 100 µg protein of cell lysates was subjected to Western blot against the antibody of FoxO1, T24, S316, and β-actin. (G and H) Blood glucose levels were measured in WT, A/+, and A/A mice at the ages 4 to 16 weeks during (G) 16 hours fasting and (H) random-fed conditions. *P < 0.05 vs WT and A/+ mice, n = 10 mice per group. M, Marker; Neo, neomycin; PGK-1, phosphoglycerate kinase 1; TK, thymidine kinase; utr, untranslated region.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Mutagenesis, Homologous Recombination, Southern Blot, DNA Sequencing, Control, Phospho-proteomics, Isolation, Western Blot, Marker

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired insulin and glucagon secretion and glucose homeostasis. (A) GIR of control and A/A mice at the ages of 16 weeks in hyperinsulinemic-euglycemic clamp assays; n = 8 mice per group. (B) HGP of mice during the clamp assay. (C and D) Glucose uptake in a variety of tissues of mice during the clamp assay. P < 0.05, n = 8 mice per group. (E) GTTs were performed in 10-week mice (n = 6) under a 16-hour fasting condition. Blood glucose was plotted against the time after the IP 2-g d-glucose/kg body weight glucose injection. (F) Serum insulin concentration was measured in mice during 16-hour fasting and random-fed states. *P < 0.05 vs WT mice, n = 10 mice per group. (G) Serum insulin concentration was measured in mice during the GTT from (E). *P < 0.05 vs WT mice, n = 10 mice per group. (H) PTTs were conducted in unfed mice after 16 hours overnight. Blood glucose levels (means ± SEM, n = 6 mice per group) were determined at the indicated time points after an IP injection of 2 g pyruvate/kg body weight. *P < 0.05 vs WT. (I) Serum glucagon concentration was measured in mice during the 16-hour fasting and random-fed states. *P < 0.05 vs WT mice, n = 10 mice per group. (J) Expression of Pck1 and G6pc in the liver of WT, A/+, and A/A under a 16-hour fasting or random-fed condition. *P < 0.05 vs WT, n = 6. Brown.ad, brown adipose; Gastro, gastrocnemius; Vastus L., vastus lateralis.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Control, Injection, Concentration Assay, Expressing

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: EE increases in FoxO1-S253A/A mice. The mice at the age of 16 weeks old were placed in metabolic cages for the measurement of food intake, physical activity, and VO2, and EE. (A) Food intake, (B) physical activity, (C) VO2, (D) EE, and (E) RER were calculated. #P < 0.05 vs WT, n = 8 mice per group.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Activity Assay

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired α-cells, insulin, and glucagon synthesis in the pancreas. (A) Representative pancreas of control (WT) and A/A mice at the age of 12 weeks at random-fed states. *P < 0.05 vs WT, n = 6. (B and C) Insulin and glucagon concentration was measured in the pancreas of control and A/A mice at the age of 12 weeks at random-fed states. *P < 0.05 vs WT, n = 3. (D and E) Insulin and glucagon concentration was measured in the islets of control and A/A mice and normalized by islet protein. *P < 0.05 vs WT, n = 3. (F) Insulin secretion from the islets isolated from control and A/A mice. *P < 0.05 vs WT, n = 3. (G) Glucagon secretion from the islets isolated from control and A/A mice under 3.3 mM glucose condition. *P < 0.05 vs WT, n = 3. (H) Immunostaining of pancreas from 16-week-old random-fed mice by glucagon and insulin. Representative images are shown. *P < 0.05 vs WT, n = 6 mice per group. (I–K) The number of (I) α-cells and (J) β-cells was counted from at least three mice per group, and the (K) ratio of α-cell/β-cell number in each islet was counted and calculated. *P < 0.05 vs WT, n = 3 mice per group. (L) The representative images of the pancreas and islets of WT and A/A mice. (M) The area of islets in total pancreas was calculated in WT and A/A mice. *P < 0.05 vs WT, n = 4. (N and O) The expression of marker genes responsible for the pancreatic β-cell, α-cell, acinar cell, and progenitor cell in (N) total pancreas or (O) islets isolated from WT and A/A mice. *P < 0.05 vs WT, n = 3. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Control, Concentration Assay, Isolation, Immunostaining, Expressing, Marker

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. *P < 0.05 vs control, n = 6 mice per group. (B) Blood glucose in random-fed mice injected by glucagon. *P < 0.05 vs control, n = 8 per group. (C) Glucagon tolerance tests in control and A/A mice at the age of 12 weeks. Blood glucose was measured in 18-hour unfed mice by 16 µg/kg body weight of glucagon IP injection. *P < 0.05 vs control, n = 8 mice per group. (D) HGP assay in primary hepatocytes of control and A/A mice. Cells were stimulated by 100 nM glucagon, and glucose concentrations in culture medium were measured. *P < 0.05 WT vs WT + glucagon; #P < 0.05 S253A/A vs S253A/A + glucagon, n = 3 experiments. (E) HGP, glycogenolysis, and gluconeogenesis were measured in the primary hepatocytes. *P < 0.05 vs WT; #P < 0.05 vs A/A, n = 3 experiments. (F) Gene-expression levels of G6pc were measured in primary hepatocytes by real-time PCR. Data presented as average of triplicate determinations from control and A/A or 100 nM glucagon stimulation for 8 hours. *P < 0.05, n = 3. PCR primer sequences are cyclophilin 5′-ctaaagcatacaggtcctggcatcttg-3′ and 5′-tgccatccagccattcagtcttg-3′; G6pc are 5′-cattgtggcttccttggtcc-3′ and 5′-ggcagtatgggataagactg-3′. (G) Nuclear (N) and cytoplasmic (C) proteins were extracted from control and A/A primary hepatocytes, with or without 100 nM glucagon (Gcg) stimulation for 30 minutes. Cytoplasmic proteins (100 µg) and 20 µg nuclear proteins were subjected to SDS-PAGE and Western blot analysis. Quantification of nuclear FoxO1 was normalized by histone-H1 with analyses from ImageJ. *P < 0.05 vs control, n = 3 experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N.S., no significance.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Gene Expression, Concentration Assay, Control, Injection, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: Dephosphorylation of FoxO1-S253 in FoxO1-S253A/A mice regulates hepatic gluconeogenesis, glycogen metabolism, and pancreatic plasticity. (A) FoxO1-S253 de-phosphorylation controls insulin and glucagon production in the pancreas, regulating liver glucose metabolism in the feeding state, resulting in an increase of blood glucose. (B) At the fasting state, where blood glucagon decreases, reducing the blood glucose. →, stimulation; ˧, inhibition.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: De-Phosphorylation Assay, Inhibition

Journal: Journal of Diabetes Research

Article Title: Sirt1 Mediates Vitamin D Deficiency-Driven Gluconeogenesis in the Liver via mTorc2/Akt Signaling

doi: 10.1155/2022/1755563

Figure Lengend Snippet: 1,25(OH) 2 D 3 deficiency leads to increased gluconeogenesis and hepatic insulin resistance through impairing phosphorylation of AKT and phosphorylation of FOXO1. (a) ITT was performed in 6-month-old 1 α (OH)ase −/− and wild-type mice ( n = 8). ns: not significant; blue line: RDWT group; red line: RDKO group; black bar: RDWT group; gray bar: RDKO group. (b, c) Insulin stimulated the phosphorylation of AKT-S473 and FOXO1-S256 in wild-type livers markedly but failed to do so in the mutant ones, as measured by Western blotting (b) and IHC (c). ∗ P < 0.05. Scale bar = 100 μ m; magnification: 20x (c). (d) The phosphorylation of AKT-S473 and FOXO1-S256 was also inhibited in HepG2 cells transfected with si-VDR compared with HepG2 cells transfected with si-NC. ∗ P < 0.05. (e) Ectopic overexpression of FOXO1 increases transcriptional activity of PCK1 promoter. This effect was enhanced by knockdown of VDR. HepG2 cells were transfected with PCK1-luc, together with a FOXO1 expression vector (FOXO1) or a GFP expression vector as a control (GFP). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Immunohistochemical staining for FOXO1 phosphorylated at S256, AKT phosphorylated at S473, and Sirt1 was carried out using the avidin-biotin-peroxidase complex technique with antibodies against FOXO1 phosphorylated at S256 (Cell Signaling Technology, #84192), AKT phosphorylated at S473 (Cell Signaling Technology, #4060), and Sirt1 (Abcam, #ab189494).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Transfection, Over Expression, Activity Assay, Knockdown, Expressing, Plasmid Preparation, Control

Journal: Journal of Diabetes Research

Article Title: Sirt1 Mediates Vitamin D Deficiency-Driven Gluconeogenesis in the Liver via mTorc2/Akt Signaling

doi: 10.1155/2022/1755563

Figure Lengend Snippet: Supplementation of 1,25(OH) 2 D 3 or overexpression of Sirt1 alleviates hepatic glucose overproduction and glucose metabolism disorders caused by 1,25(OH) 2 D 3 deficiency. (a) PTT performed in mice of different treatment groups. Blue line: RDWT group; red line: RDKO group; purple line: RD 1 α (OH)ase-/-SIRT1Tg group; green line: ND 1 α (OH)ase-/-+1,25(OH)2D3 group. (b) GTT performed in mice of different treatment groups. Blue line: RDWT group; red line: RDKO group; purple line: RD 1 α (OH)ase-/-SIRT1Tg group; green line: ND 1 α (OH)ase-/-+1,25(OH)2D3 group. (c) Expression levels of proteins including VDR, Sirt1, Rictor, PCK1, and G6PC collected from liver samples of mice of each group under normal feeding conditions. (d) Phosphorylation of AKT-S473 and FOXO1-S256 in the liver of mice stimulated with insulin. (e) VDR and Sirt1 protein expression levels in HepG2 cells treated with different concentrations of 1,25(OH) 2 D 3 or Sirt1 agonists (SRT1720HCL) were determined by Western blotting. (f) Glucose production content of HepG2 cells with knockdown of VDR or treated with 1,25(OH) 2 D 3 or SRT1720 HCL in response to pyruvate stimulation. ∗∗∗ P < 0.001; blue bar: si-NC group; red bar: si-VDR group; purple bar: 1,25(OH) 2 D 3 group; green bar: SRT1720HCL group. (g) Detection of mRNA expression of related genes in the liver of different treatment groups (qRT-PCR). ∗ P < 0.05. (h) Western blotting was performed to detect the expression levels of related proteins in different treatment groups of HepG2 cells. (i) The phosphorylation of AKT-S473 and FOXO1-S256 in HepG2 cell treated with different concentrations of 1,25(OH) 2 D 3 and Sirt1 agonist (SRT1720HCL) in response to insulin stimulation was measured by Western blotting.

Article Snippet: Immunohistochemical staining for FOXO1 phosphorylated at S256, AKT phosphorylated at S473, and Sirt1 was carried out using the avidin-biotin-peroxidase complex technique with antibodies against FOXO1 phosphorylated at S256 (Cell Signaling Technology, #84192), AKT phosphorylated at S473 (Cell Signaling Technology, #4060), and Sirt1 (Abcam, #ab189494).

Techniques: Over Expression, Expressing, Phospho-proteomics, Western Blot, Knockdown, Quantitative RT-PCR

Journal: Journal of Diabetes Research

Article Title: Sirt1 Mediates Vitamin D Deficiency-Driven Gluconeogenesis in the Liver via mTorc2/Akt Signaling

doi: 10.1155/2022/1755563

Figure Lengend Snippet: Model of mechanisms leading from 1,25(OH) 2 D 3 deficiency to increased hepatic gluconeogenesis. 1,25(OH) 2 D 3 -mediated pathway and insulin-mediated pathway converge on AKT, working together to promote AKT phosphorylation at Ser473 and then FOXO1 phosphorylation at Ser256. 1,25(OH) 2 D 3 deficiency suppresses Sirt1 expression, leading to downregulation of Rictor which further impaired phosphorylation of AKT-S473 and FOXO1-S256. As a result, the expression of G6Pase and PCK1 is upregulated, causing hepatic glucose overproduction and hepatic insulin resistance.

Article Snippet: Immunohistochemical staining for FOXO1 phosphorylated at S256, AKT phosphorylated at S473, and Sirt1 was carried out using the avidin-biotin-peroxidase complex technique with antibodies against FOXO1 phosphorylated at S256 (Cell Signaling Technology, #84192), AKT phosphorylated at S473 (Cell Signaling Technology, #4060), and Sirt1 (Abcam, #ab189494).

Techniques: Phospho-proteomics, 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: Endocrinology

Article Title: Muscle-specific overexpression of heparin-binding epidermal growth factor-like growth factor increases peripheral glucose disposal and insulin sensitivity.

doi: 10.1210/en.2008-1647

Figure Lengend Snippet: FIG. 5. Activation of Akt-FoxO signaling by HB-EGF. A, HB-EGFmTg (no. 45) or WT mice (n 2 for each genotype) at 22 wk of age were injected iv with saline or insulin at a dose of 0.2 U/kg (for analysis of Akt) or 2 U/kg (for analysis of FoxO). The mice were killed 15 min later, and lysates of gastrocnemius muscle were subjected to immunoblot analysis with antibodies specific for the Thr308- or Ser473-phosphorylated forms of Akt (Akt-p-Thr308 or Akt-p-Ser473), total Akt, the Thr24-phosphorylated form of FoxO1 (which also react with the Thr32

Article Snippet: Lysates were centrifuged at 10,000 g for 15 min at 4 C, and the resulting supernatants were subjected to immunoblot analysis with the following antibodies: polyclonal antibodies to HB-EGF generated in our laboratory by injecting rabbits with a synthetic peptide (GYDLESEEKVKLGVASSH) corresponding to the cytoplasmic region of the mouse protein; rabbit polyclonal antibodies to the Thr308- or Ser473-phosphorylated forms of Akt, to total Akt, the Thr24phosphorylated form of FoxO1 (which cross-react with the Thr32 phosphorylated form of FoxO3a), or total FoxO1 (Cell Signaling, Beverly, MA); and a mouse monoclonal antibody to -tubulin (Sigma-Aldrich).

Techniques: Activation Assay, Injection, Saline, Western Blot