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: Journal of Orthopaedic Translation

Article Title: FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

doi: 10.1016/j.jot.2024.08.010

Figure Lengend Snippet: Figure 3. FOXO1 inhibition in pericytes prevents angiogenesis. a-c. Representative images (a) and quantitative analyses (b and c) of tube-forming assays of HUVECs. HUVECs were cultured in conditional medium (CM) from HBVPs treated with various with concentrations of AS1842856 (0, 0.1, 1 μM), and collected after 8, 12 and 20 h. Scale bars represent 200 μm. n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001. d, e. Representative images (d) and wound healing rates (healed area/scratch area%) (e) in the scratch assay showing HUVECs migration under different conditional cultures. Scale bars represent 500 μm. n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001. f, g. qPCR analysis of VEGF (f) and ANG-1 (g) mRNA expression in HBVPs treated with various concentrations of AS1842856 for 48 h. n = 4/group. *P < 0.05. Data are represented as mean ± SEM and individual points represent independent wells of cultured cells. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (b, c and f) or Dunnett’s T3 test (e and g).

Article Snippet: To investigate the role of FOXO1 in bone metabolism, we crossed Col2-CreERT mice (RRID: IMSR_JAX: 006774) with Foxo1flox/+mice (Quote: TOS191231MG2-B, Stock No. 017986, Cyagen Bioscience Inc. China) to generate Col2-CreERT/Foxo1flox/flox mice.

Techniques: Inhibition, Cell Culture, Wound Healing Assay, Migration, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

doi: 10.1016/j.jot.2024.08.010

Figure Lengend Snippet: Figure 7. FOXO1 in PDGFRαþ pericytes maintains type H vessels by inhibiting mTOR signaling in mice bone. a. Representative immunofluorescence images of PDGFRα (green) and p-mTOR (red) in the femoral metaphysis of 4-week-old Adipo-Cre/Foxo1f/f and Foxo1f/f mice. Nuclei were stained DAPI. Scale bars represent 50 μm. b, c. Quantitative analysis of PDGFRα+ fluorescence intensity (b) and PDGFRα+ p-mTOR+ colocalization fluorescence intensity (c). n = 6/group. ***P < 0.001. d. Representative immunofluorescence images of PDGFRα (red) and p-mTOR (green) in 8- and 12-month-old mice treated with AS1842856. Nuclei were stained DAPI. Scale bars represent 50 μm. e-g. Quantitative analysis of PDGFRα+ (e), phosphorylated mTOR+ (f), and their colocalization (g) fluorescence signal intensities. n = 6/group. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using Student’s t-test (equal variances) (b and c) or one-way ANOVA with Tukey’s post-hoc test (equal variances) (e, f and g).

Article Snippet: To investigate the role of FOXO1 in bone metabolism, we crossed Col2-CreERT mice (RRID: IMSR_JAX: 006774) with Foxo1flox/+mice (Quote: TOS191231MG2-B, Stock No. 017986, Cyagen Bioscience Inc. China) to generate Col2-CreERT/Foxo1flox/flox mice.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Cell reports

Article Title: Combined inhibition of de novo glutathione and nucleotide biosynthesis is synthetically lethal in glioblastoma

doi: 10.1016/j.celrep.2025.115596

Figure Lengend Snippet: (A–E) TERT mRNA (A), ATRX mRNA (B), GSH pool size (C), percentage of 13 C labeling of GSH from [U- 13 C]-glutamine (D), and GCLC mRNA (E) in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (F) Western blots for GCLC in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. β-actin was used as the loading control. (G) GCL activity in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (H) Western blots for phosphorylated and total FOXO1 in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (I) FOXO1 binding to the GCLC promoter as measured by ChIP-qPCR in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (J) Western blots for the FLAG tag in NHA CONTROL , NHA TERT , and NHA TERT cells expressing a FLAG-tagged constitutively active form of FOXO1 (CA-FOXO1). β-actin was used as the loading control. (K) FOXO1 binding to the GCLC promoter measured by ChIP-qPCR in NHA CONTROL , NHA TERT , and NHA TERT cells expressing CA-FOXO1. (L–N) GCLC protein expression (L), GSH pool size (M), and percentage of 13 C labeling of GSH from [U- 13 C]-glutamine (N) in NHA CONTROL , NHA TERT , and NHA TERT cells expressing a FLAG-tagged CA-FOXO1. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns indicates lack of statistical significance. See also .

Article Snippet: Human FOXO1 primers , Origene , HP205770.

Techniques: Labeling, Control, Western Blot, Activity Assay, Binding Assay, ChIP-qPCR, FLAG-tag, Expressing, Standard Deviation

Journal: Cell reports

Article Title: Combined inhibition of de novo glutathione and nucleotide biosynthesis is synthetically lethal in glioblastoma

doi: 10.1016/j.celrep.2025.115596

Figure Lengend Snippet: (A–C) FOXO1 binding to the GCLC promoter (A), GCLC protein expression (B), and GSH pool size (C) in GBM6, U251, SB28, SF10417, A375, and HepG2 cells transfected with non-targeting control small interfering RNA (siRNA; siNT) or two non-overlapping siRNA sequences against TERT (siTERT-1 and siTERT-2). (D–F) FOXO1 binding to the GCLC promoter (D), GCLC mRNA (E), and GSH pool size (F) in BT142, KNS42, and U2OS cells transfected with an empty vector or with a plasmid expressing ATRX. (G–I) TERT mRNA (G), FOXO1 binding to the GCLC promoter (H), and GCLC mRNA (I) in GBM, astrocytoma, or gliosis biopsies. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns indicates lack of statistical significance.

Article Snippet: Human FOXO1 primers , Origene , HP205770.

Techniques: Binding Assay, Expressing, Transfection, Control, Small Interfering RNA, Plasmid Preparation, Standard Deviation

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: 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