Journal: bioRxiv

Article Title: Metabolic sensor AMPK licenses CD103 + dendritic cells to induce Treg responses

doi: 10.1101/2023.02.21.528293

Figure Lengend Snippet: (A) Gene expression of PPARγ and PGC1α in RA-treated and control moDCs. On day 5 of culture moDCs were treated either with RA or left untreated for 48 hours and mRNA expression levels were determined by RT-qPCR. GAPDH was used as housekeeping gene (B-C) . On day 5, moDCs were pre-treated with either DMSO, PPARγ (GW9662) or PGC1α (SR18292) inhibitors for 30 minutes prior to RA treatment, followed by LPS stimulation and (B) CD103 expression and (C) RALDH activity were quantified by flow cytometry. (D) In silico analysis displaying the top predicted AMPK-phosphorylation sites in FoxO3. Dashed line represents the threshold level of AMPK-FoxO3 interaction. (E) Representative immunoblot (left) and quantification (right) of the expression levels of FoxO3 Ser413, FoxO3 Ser253, total FoxO3 and β-actin in RA-DCs. (F) Representative immunoblot (left) and quantification (right) of the expression levels of FoxO3 Ser413, total FoxO3 and β -actin in RA-DCs silenced for AMPKa1 as described in . (G) Expression levels of CD103 and (H) RALDH activity in RA-DCs silenced for FoxO3 as described in . (I) moDCs were silenced for FoxO3 as described in and T cell suppression assay was performed as described in . Data are a pool of 3 (A) ; 4-8 (B-C) , 3-4 (E-F) , or 4-7 donors (G-H) or are representative for 1 out of 3 independent experiments (I) . Statistics are student’s T-test (A;E) and Two-Way Anova with Sidák’s posttest (B-C;F-I) , were used to assess statistically significant differences. Mean ± SEM are indicated in the graphs; *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.

Article Snippet: The primary antibodies used were AMPK (Cell Signaling #2532), pAMPK Ser79 (Cell Signaling #2535S), FoxO3 (Cell Signaling #2497S), pFoxO3 Ser413 (Cell Signaling #8174S), pFoxO3 Ser 253 (Cell Signaling #9466), HSP90 (Santa Cruz #sc7947) and beta-actin (Sigma #A5441).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Flow Cytometry, In Silico, Western Blot, Suppression Assay

Journal: Frontiers in Pharmacology

Article Title: Crebanine induces ROS-dependent apoptosis in human hepatocellular carcinoma cells via the AKT/FoxO3a signaling pathway

doi: 10.3389/fphar.2023.1069093

Figure Lengend Snippet: Effects of antioxidant NAC on mitochondria and mitochondria-associated proteins in crebanine-treated HepG2 cells. (A) and (B) HepG2 cells were pretreated with or without NAC for 1 h before exposure to crebanine, JC-1 staining and flow cytometry were performed to evaluate the effect of crebanine on mitochondrial membrane potential (Δψm) ( n = 3 for each group, one-way ANOVA with Tukey’s post hoc test, *** p < 0.001 vs. Control, ### p < 0.001 vs. only crebanine treated groups). (C) and (D) HepG2 cells were pre-treated with NAC, then treated with crebanine for 24 h. Cleaved PARP, caspase-3, -9, Bcl-2 and Bax levels were determined by Western blotting ( n = 3 for each group, one-way ANOVA with Tukey’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. vs. only crebanine treated groups). (E, F) Western blot was used to measure the level of signaling pathway proteins (AKT, p-Akt, FoxO3a, p-FoxO3a (Ser253)) ( n = 3 for each group, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, *** p < 0.001 vs. control). All data are expressed as mean ± SD.

Article Snippet: Crebanine with purity above 97% was provided by Chengdu pureChem-standard Corp. (Chengdu, China); Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Inc. United States); LY294002 (PI3K Inhibitor) was purchased from MedChemExpress (NJ, United States); N-acetylcysteine (NAC) was obtained from Sigma Chemical (St. Louis, MO, United States); Hoechst 32258 was obtained from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China); CCK-8 kit was purchased from Biosharp (Anhui, China); the ROS assay kit and JC-1 kit were obtained from Beyotime Biotechnology (Shanghai, China); Superoxide Dismutase (SOD) assay kit、Malondialdehyde (MDA) assay kit、Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng bioengineering research institute (Nanjing, China); Annexin V/PI apoptosis detection kit was obtained from BD Biosciences (NY, United States); the monoclonal antibody against Bax, caspase-3, Bcl-2, AKT, p-AKT, FoxO3a, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (CST, United States); the monoclonal antibody against caspase-9, p-FoxO3a (Ser253) were obtained from Proteintech (Rosmont, IL, United States).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Crebanine induces ROS-dependent apoptosis in human hepatocellular carcinoma cells via the AKT/FoxO3a signaling pathway

doi: 10.3389/fphar.2023.1069093

Figure Lengend Snippet: Crebanine induced apoptosis of HepG2 cells through ROS accumulation to regulate the Akt/FoxO3a pathway. (A) and (B) Cells were pretreated with NAC and then incubated with crebanine for 24 h. Western blot measured the protein levels of AKT, p-Akt, FoxO3a, p-FoxO3a (Ser253) ( n = 3 for each group, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, *** p < 0.001 vs. Control; ### p < 0.001 vs. only crebanine treated groups). (C) and (D) Crebanine was applied in combination with the AKT inhibitor LY294002 to the HepG2 cells to identify the AKT, p-Akt, FoxO3a, p-FoxO3a (Ser253) expression through western blot (n = 3 for each group, one-way ANOVA with Tukey’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; # p < 0.05 vs. only LY294002 treated groups). All data are expressed as mean ± SD.

Article Snippet: Crebanine with purity above 97% was provided by Chengdu pureChem-standard Corp. (Chengdu, China); Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Inc. United States); LY294002 (PI3K Inhibitor) was purchased from MedChemExpress (NJ, United States); N-acetylcysteine (NAC) was obtained from Sigma Chemical (St. Louis, MO, United States); Hoechst 32258 was obtained from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China); CCK-8 kit was purchased from Biosharp (Anhui, China); the ROS assay kit and JC-1 kit were obtained from Beyotime Biotechnology (Shanghai, China); Superoxide Dismutase (SOD) assay kit、Malondialdehyde (MDA) assay kit、Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng bioengineering research institute (Nanjing, China); Annexin V/PI apoptosis detection kit was obtained from BD Biosciences (NY, United States); the monoclonal antibody against Bax, caspase-3, Bcl-2, AKT, p-AKT, FoxO3a, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (CST, United States); the monoclonal antibody against caspase-9, p-FoxO3a (Ser253) were obtained from Proteintech (Rosmont, IL, United States).

Techniques: Incubation, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Crebanine induces ROS-dependent apoptosis in human hepatocellular carcinoma cells via the AKT/FoxO3a signaling pathway

doi: 10.3389/fphar.2023.1069093

Figure Lengend Snippet: Distribution of p-FoxO3a in HepG2 cells after crebanine action on cells. (A) Cells need to be pretreated with NAC before administration of crebanine as a way to monitor changes in p-FoxO3a expression in cells (n = 3 for each group). (B) After LY294002 pretreatment of cells, the distribution of p-FoxO3a was detected again ( n = 3 for each group).

Article Snippet: Crebanine with purity above 97% was provided by Chengdu pureChem-standard Corp. (Chengdu, China); Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Inc. United States); LY294002 (PI3K Inhibitor) was purchased from MedChemExpress (NJ, United States); N-acetylcysteine (NAC) was obtained from Sigma Chemical (St. Louis, MO, United States); Hoechst 32258 was obtained from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China); CCK-8 kit was purchased from Biosharp (Anhui, China); the ROS assay kit and JC-1 kit were obtained from Beyotime Biotechnology (Shanghai, China); Superoxide Dismutase (SOD) assay kit、Malondialdehyde (MDA) assay kit、Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng bioengineering research institute (Nanjing, China); Annexin V/PI apoptosis detection kit was obtained from BD Biosciences (NY, United States); the monoclonal antibody against Bax, caspase-3, Bcl-2, AKT, p-AKT, FoxO3a, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (CST, United States); the monoclonal antibody against caspase-9, p-FoxO3a (Ser253) were obtained from Proteintech (Rosmont, IL, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Crebanine induces ROS-dependent apoptosis in human hepatocellular carcinoma cells via the AKT/FoxO3a signaling pathway

doi: 10.3389/fphar.2023.1069093

Figure Lengend Snippet: Schematic diagram of the potential signaling pathway triggering apoptosis by crebanine in HepG2 cells. Crebanine treatment triggered the activation of mitochondrial ROS, which caused apoptosis while suppressing the activation of AKT/FoxO3a signaling pathway, further accelerating the death of HepG2 cells.

Article Snippet: Crebanine with purity above 97% was provided by Chengdu pureChem-standard Corp. (Chengdu, China); Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Inc. United States); LY294002 (PI3K Inhibitor) was purchased from MedChemExpress (NJ, United States); N-acetylcysteine (NAC) was obtained from Sigma Chemical (St. Louis, MO, United States); Hoechst 32258 was obtained from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China); CCK-8 kit was purchased from Biosharp (Anhui, China); the ROS assay kit and JC-1 kit were obtained from Beyotime Biotechnology (Shanghai, China); Superoxide Dismutase (SOD) assay kit、Malondialdehyde (MDA) assay kit、Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng bioengineering research institute (Nanjing, China); Annexin V/PI apoptosis detection kit was obtained from BD Biosciences (NY, United States); the monoclonal antibody against Bax, caspase-3, Bcl-2, AKT, p-AKT, FoxO3a, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (CST, United States); the monoclonal antibody against caspase-9, p-FoxO3a (Ser253) were obtained from Proteintech (Rosmont, IL, United States).

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway

doi: 10.3390/ijms24043807

Figure Lengend Snippet: Representative images and quantitative analysis for Akt, FoxOs and eNOS expressions in STZ-induced diabetic mice with or without febuxostat treatment. ( a ) Representative immunoblot showing Akt, FoxOs and eNOS expression levels in mouse kidneys. ( b ) Quantitative analyses for phosphor-Ser 473 Akt/total Akt, ( c ) Quantitative analyses for phospho-Ser 256 FoxO1/total-FoxO1. ( d ) Quantitative analyses for phospho-Ser 253 FoxO3a/total-FoxO3a. ( e ) Quantitative analyses for phospho-Ser 1173 eNOS/total eNOS, * p < 0.05 and *** p < 0.001 vs. Cont, †† p < 0.01 and ††† p < 0.001 vs. Feb and ‡ p < 0.05, ‡‡ p < 0.01, and ‡‡‡ p < 0.001 vs. STZ group.

Article Snippet: The membrane was incubated with primary antibodies against the following target proteins: VEGFR1 (Boster, Pleasanton, CA, USA), VEGFR2 (St John’s Laboratory, London, UK), VEGFR3 (NSJ Bioreagent Logo, San Diego, CA, USA), NOX1 and NOX4 (Lifespan Biosciences, Seattle, WA, USA), NOX2 (BD Biosciences, San Jose, CA, USA), total Akt, phospho-Ser 473 Akt, SOD1 and SOD2 (Cell Signaling Technology, Danvers, MA, USA), total FoxO1, phospho-Ser 256 FoxO1, total FoxO3a, and phospho-Ser 253 FoxO3a (Novus Biologicals, Centennial, CO, USA), total endothelial nitric oxide synthase (eNOS, EMD Millipore, Middlesex County, MA, USA), and phospho-Ser 1173 eNOS (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway

doi: 10.3390/ijms24043807

Figure Lengend Snippet: Representative images and quantitative analysis for NOX1, NOX2, NOX4, FoxO3a, and eNOS according to VEGFR1 or VEGFR3 inhibition in HG-treated human GECs with or without febuxostat. ( a ) Representative immunoblot images of NOX1, NOX2, NOX4, FoxO3a and eNOS. ( b ) Quantitative analyses of NOX1/GAPDH. ( c ) Quantitative analyses of NOX2/GAPDH. ( d ) Quantitative analyses of NOX4/GAPDH. ( e ) Quantitative analyses for phospho-Ser 253 FoxO3a/total FoxO3a. ( f ) Quantitative analyses for phospho-Ser 1173 eNOS/total eNOS. * p < 0.05, ** p < 0.01, and *** p < 0.001, Fit1: anti-Flt1 peptide, SAR: SAR131675.

Article Snippet: The membrane was incubated with primary antibodies against the following target proteins: VEGFR1 (Boster, Pleasanton, CA, USA), VEGFR2 (St John’s Laboratory, London, UK), VEGFR3 (NSJ Bioreagent Logo, San Diego, CA, USA), NOX1 and NOX4 (Lifespan Biosciences, Seattle, WA, USA), NOX2 (BD Biosciences, San Jose, CA, USA), total Akt, phospho-Ser 473 Akt, SOD1 and SOD2 (Cell Signaling Technology, Danvers, MA, USA), total FoxO1, phospho-Ser 256 FoxO1, total FoxO3a, and phospho-Ser 253 FoxO3a (Novus Biologicals, Centennial, CO, USA), total endothelial nitric oxide synthase (eNOS, EMD Millipore, Middlesex County, MA, USA), and phospho-Ser 1173 eNOS (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Characterization of placental endocrine function and fetal brain development in a mouse model of small for gestational age

doi: 10.3389/fendo.2023.1116770

Figure Lengend Snippet: List of antibodies used for western blotting.

Article Snippet: FOXO3a (D19A7) , Cell Signalling,12829 , 1/1,000.

Techniques: Western Blot

Journal: Frontiers in Endocrinology

Article Title: Characterization of placental endocrine function and fetal brain development in a mouse model of small for gestational age

doi: 10.3389/fendo.2023.1116770

Figure Lengend Snippet: List of antibodies used for western blotting.

Article Snippet: Phospho-FOXO3a (Ser318/321) , Cell Signalling, 9465 , 1/1,000.

Techniques: Western Blot

Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet: Acute treatment of H 2 O 2 induces changes in redox state of Prdx1 and Prdx2 and increased nuclear localization of redox sensitive transcription factors . Effects of different concentrations of H 2 O 2 on monomer (M)/dimer (D) ratio of Prdx1 (a) and Prdx2 (b) and Prdx SO 2 /SO 3 (c) in C2C12 myoblasts. Myoblasts were treated for 10 min with 25 μM H 2 O 2 , media was changed and % nuclear localization of transcription factors NRF2 (d), NF-κB (e), FOXO3a (f) and STAT3 (g) was analysed after 3 and 24 h; scale bar = 75 μm, n = 3–5. Protein extracts and Western blotting was performed from either Ctrl or 3 h and 24 h following H 2 O 2 treatment against PRDX1 (h), PRDX2 (i), PRDX3 (j), SOD2 (k), TRX1 (l), TRX2 (m), ULK1 (n) LC3b (o) and p62/SQSTM1 (p). Graphs are the normalized relative means ± SEM and all experiments were performed with n = 3 and p- value of <0.05 was considered as statistically significant *( p < 0.05), one-way ANOVA was used for significance between groups (a–p). p values (a: Ctrl vs 25 μM = 0.0386; b: Ctrl vs 25 μM < 0.0001; c: Ctrl vs 50 μM = 0.0277; d: Ctrl vs 3 h = 0.0066, Ctrl vs 24h = 0.0074; e: Ctrl vs 24 h < 0.0001; f: Ctrl vs 24 h < 0.0001; g: Ctrl vs 24 h < 0.0001; h: Ctrl vs 24 h = 0.0490; i: Ctrl vs 24 h = 0.0468; j: Ctrl vs 24 h = 0.0270; k: Ctrl vs 24 h = 0.0067; l: Ctrl vs 24 h = 0.0207; m: 3 h vs 24 h = 0.0355; n: Ctrl vs 24 h = 0.0128; o: Ctrl vs 3 h = 0.0091; p: Ctrl vs 24 h = 0.0061).

Article Snippet: rabbit anti-FoxO3a , Cell Signalling Technology , Cat# 2497, RRID: AB_836876.

Techniques: Western Blot

Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet:

Article Snippet: rabbit anti-FoxO3a , Cell Signalling Technology , Cat# 2497, RRID: AB_836876.

Techniques: Recombinant, Protease Inhibitor, SYBR Green Assay, Staining, Software

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: PD-1 signaling regulates expansion of lung Vγ6 + cells. (A) CD3 + T cells were isolated from the lungs of FVB/n mice and stimulated with recombinant IL-1β and IL-23 in the presence of plate-bound PD-L1-Fc or plate-bound anti-ICOS for 24 h. Supernatants were examined for IL-17A levels by ELISA. Each dot represents cells from one mouse ( n = 5–7/group). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s posthoc test; **P < 0.01, ***P < 0.001. (B) WT FVB/n mice were injected with a single dose of 200 μg anti–PD-1 or anti-ICOS followed by injections of 100 μg for two consecutive days. Control mice followed the same dosage regime with isotype control. Mice were sacrificed 24 h after the third injection. Single-cell suspensions from lung were stimulated for 3 h with PMA, ionomycin, and Brefeldin A. Cells were stained with antibodies against CD3, TCRδ, CD27, Vγ4, Vγ6, and IL-17A. The proportion of cells expressing IL-17A, the MFI of IL-17A, and the absolute number of cells is represented graphically for both lung Vγ4 + and Vγ6 + CD27 − cells. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (C) Percentage and total numbers of neutrophils in isotype control, anti–PD-1- and anti-ICOS–treated mice as measured by IDEXX ProCyte hematology analyzer. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (D) Representative Western blot analysis (from three biological replicates) of phospho-FOXO1 and total FOXO1 levels in γδ T cells from lungs of FVB/n mice stimulated as depicted. (E) CD3 + T cells were isolated from the lungs of FVB/n mice and cultured with plate-bound PD-L1-Fc for 3 h. Cells were stimulated with recombinant IL-1β and IL-23 for the last 30 min. Cells were analyzed by flow cytometry. Representative histograms are shown, and combined data are represented graphically. Each dot represents one mouse. Data are presented as mean ± SD ( n = 4 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01.

Article Snippet: Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech).

Techniques: Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Staining, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: Antibodies utilized for flow cytometry

Article Snippet: Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Cross-Species Analysis Reveals Dysthyroidism of the Ovaries as a Common Trait of Premature Ovarian Aging

doi: 10.3390/ijms24033054

Figure Lengend Snippet: Developmental and lifelong exposure to CPF in zebrafish ovaries promotes POA. ( a ) Graph represents the fertilization percentage estimated by counting the number of fertilized eggs obtained from five independent matings involving zebrafish females exposed to CPF vs. females not exposed (Vehicle). ( b ) Granulosa cell markers ( amh ) were detected by RT-qPCR. ( c – e ) OAGS genes ( dre-mir-143 , dre-mir-145, and gas5 ) were verified by RT-qPCR. ( f , g ) Representative Western blot analysis showing the level of Foxo3a/P-Foxo3a protein following CPF treatment ( n = 3/group). ( h ) Telomere length was measured from total genomic ovaries DNA by using a qPCR. Data were obtained normalizing using tubaI for mRNA, β-actin for proteins, and U6 for miRNA). Data are mean ± s.d. with five animals per group. Significant differences are indicated with * p < 0.05; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using Student’s t -test.

Article Snippet: The following antibodies were used to detect zebrafish proteins: Foxo3a (Cell signalling Tech., 2497, 1:1000), p-Foxo3a (Cell signalling Tech., 9466, 1:1000), Estrogen Receptor alpha (esr1, Santa Cruz, sc-8002, 1:1000); B-actin (Cell Signalling, 1:3000) was used to normalize data.

Techniques: Quantitative RT-PCR, Western Blot