Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Relieving colitis by miR-223 through the promotion of macrophage M2 polarization via the modulation of PPAR-γ/FOXO1 signaling. (A) Representative images of western blots indicating the protein expression levels of PPAR-γ and FOXO1. The data represent the findings from three independent experiments. (B, C) Changes in the expression of PPAR-γ and FOXO1. (D, E) Changes in the mRNA levels of PPAR-γ and FOXO1. All the data are expressed as the means ± SDs (n=6 each group). *p<0.05, **p<0.01, ***p<0.001. (F, G) Correlations of colonic miR-223 expression with PPAR-γ and FOXO1 expression in the colon. Pearson analyses were used to correlate miR-223 with colonic PPAR-γ and FOXO1 expression. PPAR-γ, peroxisome proliferator-activated receptor gamma; FOXO1, Forkhead box transcription factor O1.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Immunofluorescence analysis of FOXO1 and M1 macrophage marker CD86 in colonic tissue. Representative micrographs depict protein expression levels of FOXO1 (green) and its co-localization (yellow) with CD86 (red) in colon sections. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Immunofluorescence, Marker, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Schematic representation of the potential mechanisms underlying the targeted therapy of miR-223 supplement for DSS-induced colitis. miR-223 ameliorates DSS-induced colitis through promoting macrophage M2 polarization via modulation of PPAR-γ and FOXO1 signaling. DSS, dextran sodium sulfate.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques:

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 1. FoxO1 was predominantly expressed in ME cells. (A–D) ME cells were isolated from 8-week-old male Myh11- CreERT2/tdTomato (tdT)fl/fl mice (n = 4). Cre recombination was induced by tamoxifen injection 1 day before the assay. (A) Flow cytometry histograms. The Myh11-positive ME cells (tdT+) represented 10.6% (right) of salivary gland tissue cells without endothelial cells, hematocytes, and erythroid cells (CD31−C45−TER119−; 96.06%, left). (B,C) tdT fluorescence and FoxO1 immunofluorescence (IF) in FACS-sorted cells [crude, Myh(+), Myh(−)] (B). Bar = 20 μm. The cell population expressing FoxO1 and tdT double positive (%) in each FACS-sorted cells [crude, Myh(+), Myh(−)] (C). Threshold intensity was 30. *P < 0.05. (D) Expression of αSMA and FoxO1 in CD31−C45−TER119− cells (crude), tdT-positive [Myh(+)] and -negative [Myh(−)] cells. *P < 0.05. (E) tdT fluorescence and IF of FoxO1 and E-cadherin (E-cad) in submandibular glands (SMG) of Myh11-CreERT2/tdTfl/fl on embryonic day 16 (E16, n = 4) and at 8 weeks (8w, n = 3). The arrow head showed αSMA and FoxO1 double positive cells. Bar = 20 μm. All data were representative of three independent experiments. See also Supplementary Fig. S1.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Isolation, Injection, Flow Cytometry, Fluorescence, Immunofluorescence, Expressing

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 2. Overexpression of FoxO1 in ME cells. (A) Scatter plot of CD49f (x-axis) and EpCAM (y-axis). The cells were isolated from SMG in TP53 mutant female mice (n = 4) and analyzed by flow cytometry. EpCAMlowCD49fhigh-cells were sorted as ME cells (6.5%). (B) A schematic for integration of PiggyBac transposon vector plasmid. The Tet-On inducible gene expression system was used. FoxO1 expression was induced by doxycycline (Dox). (C) mCherry fluorescence merged with phase contrast in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 48 h. (D) Expression of FoxO1 mRNA in MEPB-FoxO1 cells treated with and without Dox for 24 h. *P < 0.05. n = 3. (E) Immunoblotting for FoxO1, αSMA, Krt14, Krt5, and β-actin in MEPB-FoxO1 cells treated with and without Dox for 72 h. (F) FoxO1 luciferase assay in the presence of FoxO1 inhibitor (Inh.; AS1842856) at the indicated concentrations. pGL4 luciferase reporter vector (upper) was constructed to include three FoxO1-binding elements (daf16:TTGTTTA and mdaf16:TTGCTTA). FoxO1 transcriptional activity was measured. pRL-TK was used as internal control. The Renilla luciferase normalized the firefly luciferase. #P < 0.05 vs. control (Ctrl). *P < 0.05 vs. Dox. n = 5. (G) Expression of αSMA mRNA in ME cells treated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) for 72 h. *P < 0.05. n = 3. (H) Expression of FoxO1 and αSMA mRNA in siRNA-mediated knockdown of FoxO1 (siFoxO1) or control (si Ctrl) in ME cells. *P < 0.05. n = 3. (I) Immunoblotting for NF-κB/p65 and phospho-NF-κB/p65 in MEPB-FoxO1 cells treated with and without Dox at the indicated time-points. The signal intensity of phospho-NF-κB/p65 was normalized to that of NF-κB/p65 (ratio). All data were representative of three independent experiments. See also Supplementary Figs. S2 and S3.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Over Expression, Isolation, Mutagenesis, Flow Cytometry, Plasmid Preparation, Gene Expression, Expressing, Fluorescence, Western Blot, Luciferase, Construct, Binding Assay, Activity Assay, Control, Knockdown

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 3. Transcriptome profiling of FoxO1-expressing ME cells. The RNA samples were prepared from MEPB-FoxO1 cells treated with and without Dox for 72 h. (A) Heatmap of differentially expressed genes (control vs. FoxO1). (B) The number of up- and down-regulated genes based on fold change of comparison pair (FoxO1/ control ≥ 2, P < 0.05). (C–F) Enrichment of Gene Ontology terms for biological processes associated with up- (C) and down- (D) regulated genes. (E,F) Data from the gene set enriched analysis (GSEA). The top gene lists of normalized enrichment score (NES) are shown in (E). *P < 0.001. Enrichment plot of cyclin A B1 B2 associated events during G2 M transition are shown in (F). See also Supplementary Tables S2–S5.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 4. FoxO1 suppressed ME cell proliferation via cell cycle arrest. (A) Viability of MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) at the indicated time-points. (B,C) Cell proliferation rates were measured by BrdU incorporation assay. BrdU positive/DAPI (%, left) with and without Dox (2 µg/mL) for 24 h (B) or with and without transfection of siRNA for FoxO1 for 48 h (C). Immunofluorescent images were showed on the right (BrdU; green, DAPI; blue). (D–F) Expression of p27(KIP1) in MEPB-FoxO1 cells. Cells were treated with and without Dox (2 µg/mL) (D), pretreated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) (E) and transfected with siRNA for FoxO1 (F) in the presence of Dox (2 µg/mL) for 48h. The expression data of p21(CIP/WAF1) were shown in Fig. S4. (G) Chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR) analysis of the DNA binding activity of FoxO1 in ME cells. DNA sample was prepared from MEPB-FoxO1 cells treated with Dox (2 µg/mL) for 72 h. The associated DNA at the promoter regions of p21CIP/WAF1 (− 1722 to − 1712) and p27KIP1 (− 1036 to − 1026), after incubation with FoxO1 antibody-conjugated protein G beads, were immunoprecipitated and analyzed by qPCR. *P < 0.05. n = 3. All data were representative of three independent experiments. (H) A schematic for FoxO1-induced cell growth inhibition. See also Supplementary Fig. S4.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: BrdU Incorporation Assay, Transfection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Activity Assay, Incubation, Immunoprecipitation, Inhibition

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 5. FoxO1 induced Eda/Eda2r expression in ME cells through NF-κB activation. (A,B) Gene expression (A) and immunoblotting (B) of Eda and Eda2r in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 24 h. (C) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) pretreatment for 24 h. (D) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without NF-κB inhibitor (MG132, 20 μM) pretreatment for 6 h. *P < 0.05. n = 3. All data were representative of three independent experiments. See also Supplementary Fig. S5.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Gene Expression, Western Blot

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 6. Inhibition of FoxO1 inhibited development of the primitive epithelium of SMG ex vivo. Epithelia of SMG rudiments on E14.5 (n = 4), were mounted in Matrigel drops and cultured in the presence of FGF1 and FGF7 with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) for 3 days. (A) Phase contrast images. Bar = 500 μm. (B) The image of immnofluorescent of αSMA, Eda, Eda2r, and phospho-NF-κB after 3 days of culture. Nuclei were stained with DAPI. Bar = 20 μm. (C) Expression of Eda and Eda2r after 3 days of culture. *P < 0.05. All data were representative of three independent experiments. See also Supplementary Figs. S6 and S7.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Cell Culture, Staining, Expressing

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 1. Rapid depletion of SOX2 and OCT4 affects the accessibility landscape of thousands of sites.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 2. Loss of SOX2 and OCT4 effect accessibility at shared and independent regions.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 5. Open chromatin regions maintained by SOX2 are associated with transcription.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The FASEB Journal

Article Title: MyD88‐mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms

doi: 10.1096/fj.202100777rr

Figure Lengend Snippet: FIGURE 4 Targeted ablation of MyD88 inhibits autophagy and FOXO transcription factors in denervated skeletal muscle. Control (sham) and 5d- or 14d-denervated GA or TA muscle of MyD88f/f and MyD88myoKO mice were analyzed by performing QRT-PCR assay and western blot. A, Relative mRNA levels of autophagy-related molecules, LC3B, Beclin1, ATG5, ATG12, and GABARAPL1 in sham-operated and 5d-denervated muscle of MyD88f/f and MyD88myoKO mice. B, Relative mRNA levels of LC3B, Beclin1, ATG5, ATG12, and GABARAPL1 in sham-operated and 14d-denervated muscle of MyD88f/f and MyD88myoKO mice. C, Representative immunoblots showing protein levels of LC3BI and LC3BII and unrelated protein GAPDH. D, Densitometry quantification of LC3BII levels in sham-operated and denervated GA of MyD88f/f and MyD88myoKO mice. E, Representative immunoblots demonstrating levels of phosphorylated and total FoxO1, FoxO3, and FoxO4 proteins in sham-operated and 5d-denerated TA muscle of MyD88f/f and MyD88myoKO mice. F, Densitometry quantification of levels of FOXO proteins in sham-operated and denervated TA muscle of MyD88f/f and MyD88myoKO mice. n = 3-6 mice per group. Data are presented as mean ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. *P < .05 (values significantly different from contralateral sham- operated TA muscle of MyD88f/f or MyD88myoKO mice). #P < .05 (values significantly different from denervated muscle of MyD88f/f mice). D, Denervated; S, sham

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 4 of 17 | PARVEEN Et Al. Biotechnology, sc- 7221), anti- phospho- FoxO1 (Thr24)/ FoxO3a (Thr32) (Cell Signaling Technology #9464), antiFoxO1 (Cell Signaling Technology #2880, anti- FoxO3a (Cell Signaling Technology #12829), anti- phospho- FoxO4 Cell Signaling Technology #9471) anti- FoxO4 (Cell Signaling Technology #2499), anti- LC3BI/II (Cell Signaling Technology, #2775), anti- Fn14 (Cell Signaling technology, #4403), anti- phospho- p65 (Cell Signaling Technology, #3033), anti- p65 (Cell Signaling Technology, #8242), antip100/52 (Cell Signaling Technology, #4882) anti- p- AMPK (Cell Signaling Technology, #2535), anti- AMPK (Cell Signaling Technology, #2532), anti- phospho- mTOR (Cell Signaling Technology, #2971), anti- mTOR (Cell Signaling Technology, #2972), anti- PERK (Cell Signaling technology, #5683), anti- ATF4 (Cell Signaling Technology, #11815), anti- CHOP (Cell Signaling Technology #2895), anti- IRE1α (Cell Signaling Technology), anti- sXBP1 (Cell Signaling Technology #12782), and anti- GAPDH (Cell Signaling Technology, #2118).

Techniques: Control, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cell reports

Article Title: Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria

doi: 10.1016/j.celrep.2023.113389

Figure Lengend Snippet:

Article Snippet: FoxO1 (D7C1H) , Cell Signaling Technology , Cat# 14952; RRID:AB_2722487.

Techniques: Virus, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Reverse Transcription, Glucose Assay, Colorimetric Assay, Mass Spectrometry, Plasmid Preparation, Software, Transfection, Blocking Assay, Protease Inhibitor

Journal: Journal of Endocrinology

Article Title: Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

doi: 10.1530/joe-13-0334

Figure Lengend Snippet: Figure 5 Insulin pretreatment attenuates insulin signal transduction in mHypoA- POMC/GFP-1 neurons. Cells were pretreated with either 100 nM insulin (C) or PBS (K) for 24 h, washed with PBS for 1 h, and then re-challenged with 10 nM insulin or PBS for 15 min. Results are normalized to levels of total protein or Gb (meansGS.E.M., nZ3–4 independent experiments). Repre- sentative blots are shown below each graph. Insulin pretreatment severely attenuates insulin-induced phosphorylation of AKT (***P!0.001 by one- way ANOVA) and of FOXO1 in mHypoA-POMC/GFP-1 (*P!0.05). Total IRb levels are also reduced by insulin pretreatment in mHypoA-POMC/GFP-1 (***P!0.001).

Article Snippet: The anti-Akt, phospho-specific-Akt (Ser473), ERK1/2 (p44/p42), phospho-specific-ERK1/2 (Thr202/Tyr204), IRb, antiFOXO1, and phospho-specific-FOXO1 (Ser256) antibodies were obtained from Cell Signaling Technology, Inc.

Techniques: Transduction, Phospho-proteomics

Journal: Cell reports

Article Title: Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria

doi: 10.1016/j.celrep.2023.113389

Figure Lengend Snippet:

Article Snippet: Phospho-FoxO1 (Ser256) Rabbit mAb , Cell Signaling Technology , Cat# 84192; RRID:AB_2800035.

Techniques: Virus, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Reverse Transcription, Glucose Assay, Colorimetric Assay, Mass Spectrometry, Plasmid Preparation, Software, Transfection, Blocking Assay, Protease Inhibitor