Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: The effects of CAT on CD4 + IL-17 + Th17 cells and the expression of let-7g-5p in CIA mice. (A) The percentage of CD4 + IL-17 + Th17 cells in PBMCs of CIA mice. (B) The percentage of CD4 + IL-17 + Th17 cells in splenocytes from CIA mice. (C) Proportion of CD4 + IL-17 + Th17 cells in PBMCs from CIA mice. (D) Proportion of CD4 + IL-17 + Th17 cells in spleens from CIA mice. (E) IL-17 levels in plasma from CIA mice were measured by ELISA. (F) The mRNA levels of RORγt in splenic CD4 + T cells were detected by qPCR. (G) The mRNA levels of let-7g-5p in splenic CD4 + T cells were detected by qPCR. Values are presented as a mean±SEM (n=6). * p <0.5, ** p <0.01, *** p <0.001 compared with the model group. CAT, catalpol; CIA, collagen-induced arthritis; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; PBMC, peripheral blood mononuclear cells; IL, interleukin.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses the differentiation of Th17 cells in vitro. (A) Representative flow cytometric pictures indicate the percentage of CD4 + IL-17 + Th17 cells differentiated from naïve CD4 + T cells. (B) The proportion of CD4 + IL-17 + Th17 cells in traditional differentiation of Th17 cells. (C) IL-17 levels in culture supernatants during traditional differentiation of Th17 cells were measured by ELISA. (D) The mRNA levels of RORγ t during traditional differentiation of Th17 cells were detected by qPCR. (E) Representative flow cytometric pictures indicate the percentage of CD4 + IL-17 + Th17 cells differentiated from Tregs. (F) The proportion of CD4 + IL-17 + Th17 cells in Tregs-to-Th17 cell transdifferentiation. (G) IL-17 levels in culture supernatants during Tregsto-Th17 cell transdifferentiation were measured by ELISA. (H) mRNA levels of RORγ t in Tregs-to-Th17 cell transdifferentiation were detected by qPCR. Values are presented as a mean±SEM (n=3). † p <0.5, †† p <0.01, ††† p <0.001 compared with the control group. CAT, catalpol; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: Expression of let-7g-5p in traditional differentiation of Th17 cells and Tregs-to-Th17 cell transdifferentiation in vitro. (A) mRNA levels of let-7g-5p in traditional differentiation of Th17 cells were detected by qPCR. (B) mRNA levels of let-7g-5p in Tregs-to-Th17 cell transdifferentiation were detected by qPCR. Values are presented as a mean±SEM (n=3). ††† p <0.001 compared with the control group. CAT, catalpol; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Expressing, In Vitro, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: Suppression of let-7g-5p on Tregs-to-Th17 cells trans-differentiation in vitro. (A) Representative dot plots of CD4 + IL-17 + Th17 cells differentiated from Tregs in vitro. (B) Proportion of CD4 + IL-17 + Th17 cells. (C) Levels of IL-17 in culture supernatants were measured by ELISA. (D) mRNA levels of IL-17 were detected by qPCR. (E) mRNA levels of RORγ t were detected by qPCR. † p <0.5, †† p <0.01, ††† p <0.001 compared with the control group. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses Tregs-to-Th17 cell transdifferentiation by up-regulating let-7g-5p in vitro. (A) Representative dot plots of CD4 + IL-17 + Th17 cells in vitro. (B) Proportion of CD4 + IL-17 + Th17 cells after let-7g-5p mimics transfection. (C) Proportion of CD4 + IL-17 + Th17 cells after let-7g-5p inhibitor transfection. (D) IL-17 levels in culture supernatants after let-7g-5p mimics transfection were measured by ELISA. (E) mRNA levels of RORγ t after let-7g-5p mimics transfection were detected by qPCR. (F) mRNA levels of IL-17 after let-7g-5p mimics transfection were detected by qPCR. (G) IL-17 levels in culture supernatants after let-7g-5p inhibitor transfection were measured by ELISA. (H) mRNA levels of RORγ t after let-7g-5p inhibitor transfection were detected by qPCR. (I) mRNA levels of IL-17 after let-7g-5p inhibitor transfection were detected by qPCR. Values are presented as a mean±SEM (n=3). * p <0.5, ** p <0.01, *** p <0.001 compared with the model group. CAT, catalpol; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses Tregs-to-Th17 cell transdifferentiation in relation to STAT3. (A) mRNA levels of STAT3 after let-7g-5p mimics transfection were detected by qPCR. (B) mRNA levels of STAT3 after let-7g-5p inhibitor transfection were detected by qPCR. (C) Protein levels of STAT3 after let-7g-5p mimics transfection were analyzed and quantified. (D) Protein levels of STAT3 after let-7g-5p mimics transfection were analyzed and quantified. (E) Expression of STAT3 was detected by Western blot. Values are presented as a mean±SEM (n=3). * p <0.05, *** p <0.001 compared with the model group. CAT, catalpol; qPCR, quantitative polymerase chain reaction; IL, interleukin.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK is highly expressed in and required for Th17 cells. (A) Naïve CD4 T cells were cultured under each subset differentiation conditions for 3 days. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were introduced with a control empty vector (control) or a CRE recombinase-expressing vector (RV-Cre) to induce Fak deletion and cultured under Th17-polarizing conditions for 3 days (B) or various subset-polarizing conditions (C) for 3 days. (B) GFP+ cells were sorted. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (C) The expression of GFP, IFN-γ, IL-4, IL-17A, and FOXP3 was analyzed by flow cytometry (top). The statistical analysis was performed on pooled data from five independent experiments (bottom). Error bars represent the standard deviation. The significance of differences between groups was determined by one-way ANOVA (A) and Student t test (B, C) . ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK affects Th17 cell differentiation program. (A, B) Naïve CD4 T cells from Fak fl/fl mice were cultured and sorted as Figure 1B . (A) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (B) Transcript levels of Il17a , Rorc , Il23r , and Foxp3 were measured by RT-qPCR. (C, D) Naïve CD4 T cells were introduced with either the control vector (MSCV-LMP) or Fak shRNA vectors (#1, #2, and #3) and cultured under Th17-polarizing conditions for 3 days. (C) The transcript level of Fak was measured by RT-qPCR (left) and protein level of FAK was measured by immunoblot analysis (right). (D) IL-17A+ cells among the vector-transduced cells (GFP+) were measured by flow cytometry (left). GFP+ cells were sorted and the transcript level of Il17a was measured by RT-qPCR (right). All of RT-qPCR data were normalized to Gapdh . (E–G) Naïve CD4 T cells were transduced with control or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and subjected to RNA-seq analysis. (E) Scatter plot of RNA-seq data. (F) Gene ontology analysis of differentially expressed genes (DEGs) from control and RV-Cre-transduced Th17 cells. (G) Heatmap of immune/inflammatory response-related genes among the DEGs from control and RV-Cre-transduced Th17 cells. All of RT-qPCR data were normalized to Gapdh . Data in (A–D) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Plasmid Preparation, shRNA, Western Blot, Transduction, RNA Sequencing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK deficiency ameliorates the severity of EAE. (A, B) Naïve CD4 T cells from WT and Fak fl/fl Rorc cre mice were cultured under polarizing conditions toward each different subset for 3 days. Transcript levels of Ifng for Th1 cells, Il4 for Th2 cells, Il17a for Th17 cells, and Foxp3 for Treg cells were measured by RT-qPCR (A) and the percentage of IL-17A+ cells under Th17-polarizing conditions was measured by flow cytometry (B) . (C) EAE was induced in control (WT, n = 10) and Fak fl/fl Rorc cre (n = 10) mice as described in the Materials and Methods section. The symptoms of EAE were monitored every day and clinical scores were evaluated after EAE induction. (D, E) Histopathological analysis of lumbar spinal cords from control and Fak fl/fl Rorc cre mice at the peak of the disease. (D) H&E stained sections of spinal cords. Arrows indicate the inflammatory foci. (E) Immunohistochemical staining of myelin basic protein (MBP). Arrows indicate the more preserved myelin in Fak fl/fl Rorc cre mice compared with control mice. (F) IL-17A+ and IFNγ+ cells (left) and FOXP3+ cells (right) among CNS-infiltrating CD4 T cells were measured by flow cytometry. (G–J) The percentage and absolute number of CNS-infiltrating mononuclear cells were measured. (G) CD4+ cells, (H) IL-17A+ cells, (I) IFNγ+ cells, and (J) FOXP3+ cells. (K) Transcript levels of Il17a , Rorc , Il23r , Ifng , and Foxp3 in CNS-infiltrating mononuclear cells were measured by RT-qPCR. Data were normalized to Gapdh . Data in (F–K) are pooled from ten independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Control, Staining, Immunohistochemical staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates the STAT3 signaling pathway in Th17 cells. (A) Naïve CD4 T cells were cultured under various differentiation conditions for the indicated time periods and the transcript levels of Fak , Il17a , Rorc , and Il17f were measured by RT-qPCR. (B) Naïve CD4 T cells were introduced with control vector or Rorc -expressing vector ( Rorc O/X) and cultured in Th17-polarizing conditions for 3 days. Transcript levels of Rorc , Il17a , and Fak were measured by RT-qPCR. (C) Naïve CD4 T cells were cultured under Th17-polarizing conditions with dose-dependent treatment of GSK805 for 3 days. Transcript levels of Il17a and Fak were measured by RT-qPCR. (D, E) Naïve CD4 T cells from Fak fl/fl mice were cultured as described in Figure 1B and GFP+ cells were sorted. (D) Each protein level was measured by immunoblot analysis (left) and the ratios of pSTAT3/STAT3 and pSTAT5/STAT5 were calculated by densitometry (right). (E) Transcript level of Il2 was measured by RT-qPCR (left) and the protein level of IL-2 from the supernatants was measured by ELISA (right). All of RT-qPCR data were normalized to Gapdh . Data in (A, B, D, E) were pooled from three independent experiments, and data in (C) from five. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates Th17 cell differentiation through the NF-κB pathway. (A) Naïve CD4 T cells from Fak fl/fl mice were transduced with control vector or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and rested in normal media for 2 days and serum-free medium for an additional 8 hours. The cells were restimulated with anti-CD3/CD28 antibodies for the indicated time periods, and nuclear/cytoplasmic extracts were prepared. Each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were transduced with RV-Cre and cultured under Th17-polarizing conditions for 3 days (B) and 16 hours (C) and GFP+ cells were sorted. (B) Transcript level of Socs3 was measured by RT-qPCR. (C) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were reacted with an anti-RelA antibody, and precipitated DNA fragments were measured by qPCR. Isotype-matching IgG was used as a negative control. (D–F) Il17a promoter activity was measured through luciferase assay. (D) EL4 cells were transfected with pGL3- Il17a promoter vector and control or Fak siRNA, and the cells were rested for 20 hours. After transfection, the cells were divided into non-stimulated or stimulated groups, with the stimulated groups received 4 hour stimulation with PMA/ionomycin. (E) EL4 cells were transfected with the pGL3- Il17a promoter vector and rested for 20 hours with or without PDTC treatment (1 μM). (F) EL4 cells were transfected as described in (D) and rested for 20 hours with 1 μM PDTC treatment. (G–I) Naïve CD4 T cells from Rela fl/fl mice were introduced with a control empty vector (WT) or a Cre recombinase expressing vector (p65 KO) to induce Rela deletion and cultured under Th17-polarizing conditions for 3 days. (G) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (H) GFP+ cells were sorted and transcript level of Rela , Il17a , Rorc , and Il23r were measured by RT-qPCR. (I) Naïve CD4 T cells from Rela fl/fl mice were cultured as described in (G) and additionally treated with vehicle (control) or FAK inhibitor (PND1186, 1 μM). IL-17A+ and FOXP3+ cells were measured by flow cytometry. RT-qPCR data in (B, H) were normalized to Gapdh . Data in (A–I) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Transduction, Control, Plasmid Preparation, Cell Culture, Western Blot, Quantitative RT-PCR, Binding Assay, Negative Control, Activity Assay, Luciferase, Transfection, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: A FAK inhibitor blocks differentiation of Th17 cells in vitro. (A–E) Naïve CD4 T cells were cultured under Th17- or Treg-polarizing conditions with dose-dependent treatment of PND1186 for 3 days. IL-17A+ and FOXP3+ cells were measured by flow cytometry (A) and transcript levels of Il17a and Foxp3 were measured by RT-qPCR (C) in Th17 cells. FOXP3+ cells were measured by flow cytometry (B) and transcript level of Foxp3 was measured by RT-qPCR (D) in Treg cells. (E–G) Naïve CD4 T cells were cultured under Th17-polarizing conditions with vehicle (control) or PND1186 (1 μM) treatment for 3 days (E, G) and the indicated time periods (F) . (E) pSTAT3 and pSTAT5 levels were measured by flow cytometry. (F) Nuclear or cytoplasmic extracts were prepared from cultured cells, and each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (G) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were incubated with an anti-RelA antibody and the precipitated DNA fragments were measured by qPCR. An isotype-matching IgG was used as a negative control. RT-qPCR data in (C, D) were normalized to Gapdh . Data in (A–G) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot, Binding Assay, Incubation, Negative Control, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 1. TH17 cells derived in vitro show different metabolic states. (A) RT-PCR analysis of key glycolytic pathway genes in TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; gene expression was normalized to Actb. (B) ECAR of TH17 (b) and TH17 (23) cells differentiated for 96 h assessed by a glycolytic stress test. ECAR was measured under basal conditions and in response to glucose (10 mM), oligomycin (1.0 mM), and 2-deoxyglucose (2-DG) (50 mM). (C) Principal component analysis (PCA) analysis of identified metabolites of TH17 (b) and TH17 (23) cells (n = 5) differentiated in vitro for 48 h by metabolomics. (D) Metabolomics analysis of TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; the top differentially observed metabolites are shown in heat map. Data are representative of three independent experiments (A and B). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 2. TH17 cells show differential metabolic pathway gene activation in vivo. (A) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with differential TH17 signature genes highlighted (black dot). differentially-expressed genes are filtered as false discovery rate (FDR) , 0.05 and FC $ 1.5 from DESeq2. (B) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glycolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (C) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glutaminolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (D) CNS TH17 and ileum TH17 cells depend on distinct metabolic pathways. Numbers indicate the differentially expressed genes and total genes for each selected pathway. (E) KEGG pathway enrichment for CNS TH17 cell highly expressed genes. (F) KEGG pathway enrichment for ileum TH17 cell highly expressed genes. Color represents log-transferred FDR, and dot size represents the number of differentially expressed genes observed for this pathway (E and F).

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Activation Assay, In Vivo, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 3. TH17 cells derived in vitro show discrete chromatin states. (A) Scatter plot for differentially opened/closed regions (OCRs) between TH17 (23) (red) and TH17 (b) cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between TH17 (23) and TH17 (b) cells. (C) The enriched transcription factor motifs identified from top 1000 differentially OCRs between TH17 (23) cells and TH17 (b) cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals for key glycolytic genes in TH17 (23) and TH17 (b) cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Labeling, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 4. TH17 cells show discrete chromatin states in vivo. (A) Scatter plot for differentially opened/closed regions (OCRs) between CNS TH17 (red) and ileum TH17 cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between CNS TH17 and ileum TH17 cells. (C) The enriched transcription factor motifs identified from top 1000 differential OCRs between CNS TH17 cells and ileum TH17 cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals with corresponding gene expression levels for key TH17 signature genes and metabolic genes in CNS TH17 and ileum TH17 cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: In Vivo, Labeling, Gene Expression, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 5. Identification of a miR-21–Peli1–c-Rel pathway controlling pathogenic TH17 cell glycolysis. (A) WashU Epigenome Browser visualization of ATAC-seq signals across selected microRNA genome loci in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells. (B) Relative expression of microRNA in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells (n = 3). (C) KEGG pathway enrichment for genes downregulated in miR-212/2

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 6. Pharmaceutical inhibition c-Rel–mediated glycolysis in pathogenic TH17 cells prevents autoimmunity. (A) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (B) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (C) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (D) Normalized RT-PCR results for key glycolytic pathway genes in TH17 (23) cells differentiated in vitro for 48 h, treated with H2O or 500 mg/ml PTXF for 16 h. (E) ECAR of TH17 (23) cells differentiated for 48 h and then were treated with H2O or 500 mg/ml PTXF for 16 h, assessed by a glycolysis stress test. (F) Basal and maximal glycolytic capability of TH17 (23) cells differentiated for 48 h and then were treated with H2O or PTXF for 16 h. (G) EAE development in C57BL/6 mice, i.p. PBS or 100 mg/kg PTXF per mouse (n = 6) from day 7 to day 14. (H) EAE development in recipient C57BL/6 mice (sublethal irradiation; n = 6) i.p. with pathogenic TH17 (23) cells differentiated from naive 2D2 CD4+ T cells for 96 h and then treated with H2O or 500 mg/ml PTXF for 16 h. Data shown are one experiment representative of three independent experiments (A–C, G, and H). Data are representative of three independent experiments (D–F). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Irradiation, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Image of the intestine of Kaede mice before and after ex vivo photoconversion of the dissected organ by exposure to a 390 nm wavelength light for 2 minutes. (B) Representative flow cytometric analysis of T cells harvested from photoactivated (PA) PPs and BM of Kaede mice subjected or not subjected to in vivo photoconversion. Plots show the relative frequency of KaedeR total T cells, TNF+ T cells, and Th17 cells. Ten-week-old female SFB+ Kaede mice were subjected to surgical laparotomy to access the PPs in the distal SI. PP cells were photoconverted by exposing them to a 390 nm light for 2 minutes. Mice were sacrificed immediately after the photoconversion. (C–E) Relative frequency of KaedeR total T cells, TNF+ T cells, and Th17 cells in PPs from sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (F and G) Relative and absolute frequency of KaedeR total T cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (H and I) Relative and absolute frequency of KaedeR total TNF+ T cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (J and K) Relative and absolute frequency of KaedeR total Th17 cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. For panels C–K, 10-week-old Kaede mice were subjected to either ovx or sham surgery. After 2 weeks, mice underwent surgical laparotomy and PP cells were photoconverted. Mice were sacrificed 24 or 48 hours later and the number of KaedeR T cells in PPs and BM measured by flow cytometry. n = 6–14 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: Ex Vivo, In Vivo, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Representative flow cytometry plot and frequency of EGFP+ Th17 cells in the BM of WT and Tnf–/– sham-operated mice and mice that had undergone ovx. (B) Relative and absolute frequency of EGFP+ Th17 cells in the BM of WT and Tnf–/– mice subjected to sham surgery or ovx 2 weeks before adoptive transfer of IL-17A-EGFP+ cells. (C) BM CD4+ T cell EGFP MFI. In these experiments, EGFP+CD4+ Th17 cells were injected i.v. into WT and Tnf–/– mice that had been subjected to sham operation or ovx 14 days before the T cell transfer. Twenty-four hours after transfer, EGFP+CD4+ T cells (EGFP+ Th17 cells) were enumerated by flow cytometry in BM of recipient mice. (D) Relative frequency of BM Vβ14+ Th17 cells in WT and Tnf–/– mice. (E and F) Relative and absolute frequency of BM of total Th17 cells in WT and Tnf–/– mice. (G) BM Ccl20 transcript levels in Tnf–/– mice. (H) Relative frequency of BM Vβ14+ Th17 cells in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. (I and J) Relative and absolute frequency of BM of total Th17 cells in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. (K) BM Ccl20 transcript levels in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. n = 5–6 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: Flow Cytometry, Adoptive Transfer Assay, Injection

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the number of PP and BM TNF+ T cells and on the level of Tnf transcripts in mice treated with FTY720. (B) Effects of ovx on the number of PP and BM Th17 cells and on the level of Il17a transcripts in mice treated with FTY720. (C) Effects of ovx on BV/TV, Tb.Th, Tb.N, and Tb.Sp in mice treated with FTY720. (D) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp in mice treated with FTY720. (E) Effects of ovx on serum CTX levels and serum osteocalcin levels in mice treated with FTY720. (F) Effects of ovx on femoral Ct.Ar and Ct.Th in mice treated with FTY720. n = 10 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the frequency of BM Th17 cells and Vβ14+ Th17 cells and on the level of Il17a transcripts. (B) Effects of ovx on the frequency of BM TNF+IL-17+ T cells. (C) Effects of ovx on the number of BM TNF+ T cells and on the level of Tnf transcripts. (D) Effects of ovx on femoral BV/TV, Tb.Th, Tb.N, and Tb.Sp. (E) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp. (F) Effects of ovx on serum CTX levels and serum osteocalcin levels. (G) Effects of ovx on femoral Ct.Ar and Ct.Th. Mice were treated with anti-CCL20 Ab or irrelevant (Irr.) Ab. n = 5 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the number of BM TNF+ T cells and on the level of Tnf transcripts in WT mice and Cxcr3–/– mice. (B) Effects of ovx on the BM cell transcript levels of Ccl20 in WT mice and Cxcr3–/– mice. (C) Effects of ovx on the number of BM Th17 cells and on the levels of Il17a transcripts in WT mice and Cxcr3–/– mice. (D) Effects of ovx on femoral BV/TV, Tb.Th, Tb.N, and Tb.Sp in WT mice and Cxcr3–/– mice. (E) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp in WT mice and Cxcr3–/– mice. (F) Effects of ovx on serum CTX levels and serum osteocalcin levels in WT mice and Cxcr3–/– mice. (G) Effects of ovx on femoral Ct.Ar and Ct.Th in WT mice and Cxcr3–/– mice. n = 5 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: