Journal: Cell Death & Disease

Article Title: IL-27 triggers IL-10 production in Th17 cells via a c-Maf/ROR γ t/Blimp-1 signal to promote the progression of endometriosis

doi: 10.1038/cddis.2017.95

Figure Lengend Snippet: IL-27 induces high levels of C-Maf and Blimp-1 and a low level of ROR γ t in Th17 cells. ( a–c ) The mouse naive T cells ( n =6) were differentiated to Th1 (stimulation with rhIL-2: 10 ng/ml; α CD3: 10 μ g/ml; α CD28: 2 μ g/ml; rhIFN- γ : 10 ng/ml; rhIL-12: 10 ng/ml; α IL-4: 10 μ g/ml), Th2 (rhIL-2: 10 ng/ml; α CD3: 10 μ g/ml; α CD28: 2 μ g/ml; IL-4: 10 ng/ml; α IFN- γ : 10 μ g/ml; α IL-12: 10 μ g/ml), Treg (rhIL-2: 10 ng/ml; α CD3: 10 μ g/ml; α CD28: 2 μ g/ml; rhTGF- β : 30 ng/ml) or Th17 (rhIL-2: 10 ng/ml; α CD3: 10 μ g/ml; α CD28: 2 μ g/ml; rhTGF- β : 2 ng/ml; rhIL-6: 50 ng/ml) cells for 5 days in vitro , and then the MFI of Blimp-1, c-Maf and ROR γ t in these cells was analyzed by flow cytometry (one-way ANOVA). ( d ) Human naive T cells ( n =5) were differentiated into Th17 cells (rhIL-6: 50 ng/ml; rhTGF- β : 2 ng/ml; α CD3: 10 μ g/ml; α CD28: 2 μ g/ml; α IFN- γ : 10 μ g/ml; α IL-4: 10 μ g/ml; α IL-12: 10 μ g/ml) and stimulated with or without rhIL-27 (25 ng/ml) for 5 days. Then, the mRNA level of PRDM1 , MAF , RORc and IL-10 in Th17 cells was analyzed by real-time PCR. IL-27: rhIL-27 (one-way ANOVA). ( e and f ) Mouse naive T cells ( n =6) were differentiated into Th17 cells and stimulated with or without rmIL-27 (25 ng/ml) in vitro for 5 days. Then, the MFI level of Blimp-1, c-Maf and ROR γ t in Th17 cells was analyzed by flow cytometry. IL-27: rmIL-27 (Student's t -test). ( g and h ) The level of Blimp-1, c-Maf and ROR γ t in CD4 + T cells ( n =10) from the PF of mice by flow cytometry. The mouse endometriosis model was constructed according to the procedure of (Student's t -test). The data are expressed as the mean±S.E.M. * P <0.05, ** P <0.01 and *** P <0.001; # P <0.05, ## P <0.01 versus Th17 group

Article Snippet: According to the standard procedure, immunoprecipitation and immunoblotting were performed to analyze the combination of c-Maf (1:500, sc-7866, Santa Cruz, Dallas, TX, USA), Blimp-1 (1:500, sc-47732, Santa Cruz) and ROR γ (1:500 sc-81371, Santa Cruz).

Techniques: In Vitro, Flow Cytometry, Real-time Polymerase Chain Reaction, Construct

Journal: Cell Death & Disease

Article Title: IL-27 triggers IL-10 production in Th17 cells via a c-Maf/ROR γ t/Blimp-1 signal to promote the progression of endometriosis

doi: 10.1038/cddis.2017.95

Figure Lengend Snippet: The c-Maf/ROR γ t/Blimp-1 complex is involved in IL-27-triggered IL-10 + Th17 cells. ( a ) The relative luciferase activity of MAF , RORc , PRDM1 , IL-17A , IL-10 and Foxp3 in HEK-293T cells was analyzed by a dual luciferase reporter assay, after co-transfection with overexpression plasmids (pIRES2-IL-27 plasmid, CMV-MCS-C-Maf-3Flag, CMV-MCS- ROR γ t-3Flag or mock plasmids) and luciferase reporter plasmids (PGL3-MAF-Luc, PGL3-RORc-Luc, PGL3-PRDM1-Luc, PGL3-IL-17A-Luc, PGL3-IL-10-Luc or PGL3-Foxp3-Luc). The data are expressed as the mean±S.E.M. * P <0.05, ** P <0.01 and *** P <0.001 (Student's t -test). ( b and c ) The combination between Blimp-1, c-Maf and ROR γ t in HEK-293T cells was detected by co-IP assay. Flag-ROR γ , transfection with CMV-MCS-ROR γ t-3Flag plasmids; Myc-Blimp-1, transfection with Blimp-1-Myc plasmids. ( d ) The schematic role of IL-27 in IL-10 + Th17 differentiation

Article Snippet: According to the standard procedure, immunoprecipitation and immunoblotting were performed to analyze the combination of c-Maf (1:500, sc-7866, Santa Cruz, Dallas, TX, USA), Blimp-1 (1:500, sc-47732, Santa Cruz) and ROR γ (1:500 sc-81371, Santa Cruz).

Techniques: Luciferase, Activity Assay, Reporter Assay, Cotransfection, Over Expression, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection

Journal: The Journal of Reproduction and Development

Article Title: Intrauterine administration of peripheral blood mononuclear cells (PBMCs) improves embryo implantation in mice by regulating local Treg/Th17 cell balance

doi: 10.1262/jrd.2021-006

Figure Lengend Snippet: Immunofluorescence analysis of Foxp3 and RORγt in mouse uterus on Pd4.0. (A) Immunofluorescence staining in each group. Foxp3 and RORγt were stained with green and red fluorescence, respectively and the nuclei were counterstained blue. The white triangle marks the protein expression of Foxp3 and the white arrow marks the protein expression of RORγt (bar = 50 μm). (B) Three fields were randomly selected from each slice in each group for analysis. Analysis of positive integrated optical density (IOD) using Image-pro Plus 6.0 software. The results are presented as the mean ± SD. ** P < 0.01 vs . control group, # P < 0.05 vs . EID group. ## P < 0.01 vs . EID group.

Article Snippet: The membranes were incubated in TBST (50 mM Tris–HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20 [G5058; Xavier Biotechnology Co., Ltd., China]) containing 5% skimmed milk at room temperature (25°C) for 1 h and subsequently incubated with anti-Foxp3 antibody (Abcam, ab215206; 1:1,000), anti-RORγt polyclonal antibody (Biorbyt, Cambridge, Britain, orb385620; 1:1,000), or anti-GAPDH antibody (Abcam, ab37168; 1:7,000) overnight at 4°C.

Techniques: Staining, Fluorescence, Expressing, Software, Control

Journal: The Journal of Reproduction and Development

Article Title: Intrauterine administration of peripheral blood mononuclear cells (PBMCs) improves embryo implantation in mice by regulating local Treg/Th17 cell balance

doi: 10.1262/jrd.2021-006

Figure Lengend Snippet: Expression of transcriptional factors and cytokines involved in Treg and Th17 cell differentiation and function in mouse uterus on Pd4.0 and Pd7.5. Twelve mice were tested in each group. (A–C) Protein levels of Foxp3 and RORγt were determined by western blotting. (D–I) mRNA levels of Foxp3 , RORγt , IL-10 , IL-17 , IL-21 , and IL-22 were determined by RT-PCR. The data are presented as the mean ± SEM. * P < 0.05 vs . control group, # P < 0.05 vs . EID group.

Article Snippet: The membranes were incubated in TBST (50 mM Tris–HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20 [G5058; Xavier Biotechnology Co., Ltd., China]) containing 5% skimmed milk at room temperature (25°C) for 1 h and subsequently incubated with anti-Foxp3 antibody (Abcam, ab215206; 1:1,000), anti-RORγt polyclonal antibody (Biorbyt, Cambridge, Britain, orb385620; 1:1,000), or anti-GAPDH antibody (Abcam, ab37168; 1:7,000) overnight at 4°C.

Techniques: Expressing, Cell Differentiation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Frontiers in Pharmacology

Article Title: Periplanta americana extract regulates the Th17/Treg cell balance via Notch1 in ulcerative colitis

doi: 10.3389/fphar.2024.1534772

Figure Lengend Snippet: PAE regulates the balance of Th17/Treg cells in UC mice. (A) Flow cytometry analysis of Th17 (CD4 + IL-17A + ) and Treg (CD4 + CD25 + Foxp3 + ) cells in spleen. (B) Western blot images of RORγt and Foxp3 in the colon. (C, D) Analysis of gray-scale of RORγt and Foxp3 protein expression. (E) Levels of TGF-β in peripheral blood. (F) Immunofluorescence images of RORγt and IL-17A in colon (×100). (n = 4–6, * P < 0.05 and ** P < 0.01 versus control group, # P < 0.05 and ## P < 0.01 versus model group).

Article Snippet: After blocking with 5% skim milk for 1.5 h (h) at room temperature, the membranes were incubated overnight at 4°C with different primary antibodies against Occludin (13409-1-AP, Proteintech), Claudin 1 (13050-1-A, Proteintech), ZO1 (TA5145F, Abmart; 21773-1-A, Proteintech), Foxp3 (Ab215206, Abcam), RORγt (13205-1-AP, Proteintech), Math1 (21215-1-A, Proteintech), Notch1 (T55244S, Abmart; 20687-1-AP, Proteintech), Hes1(11988S, CST) and β-actin (ab179467; Abcam).

Techniques: Flow Cytometry, Western Blot, Expressing, Immunofluorescence, Control

Journal: Frontiers in Pharmacology

Article Title: Periplanta americana extract regulates the Th17/Treg cell balance via Notch1 in ulcerative colitis

doi: 10.3389/fphar.2024.1534772

Figure Lengend Snippet: PAE attenuates the disruption of tight junction proteins in HCoEpic cells by regulating activated Notch1-mediated Th17/Treg imbalance in vitro . (A) Effect of VPA on the viability of Jurkat T cells analyzed with CCK8 assay. (B) Effect of PAE on the viability of Jurkat cells analyzed with CCK-8 assay. (C) Western blot images of Notch1, Math1, RORγt, and Foxp3 in Jurkat T cells. (D–G) Analysis of gray-scale of Notch1, Math1, RORγt, and Foxp3 protein expression. (H) After notch1-activated Jurkat T cells were co-cultured with HCoEpic cells, Western blot images of occluding, claudin1, and ZO1 in HCoEpic cells. (I–K) Analysis of gray-scale of occluding, claudin1, and ZO1 protein expression. (n = 5–8, * P < 0.05 and ** P < 0.01 versus control group, # P < 0.05 and ## P < 0.01 versus VPA group).

Article Snippet: After blocking with 5% skim milk for 1.5 h (h) at room temperature, the membranes were incubated overnight at 4°C with different primary antibodies against Occludin (13409-1-AP, Proteintech), Claudin 1 (13050-1-A, Proteintech), ZO1 (TA5145F, Abmart; 21773-1-A, Proteintech), Foxp3 (Ab215206, Abcam), RORγt (13205-1-AP, Proteintech), Math1 (21215-1-A, Proteintech), Notch1 (T55244S, Abmart; 20687-1-AP, Proteintech), Hes1(11988S, CST) and β-actin (ab179467; Abcam).

Techniques: Disruption, In Vitro, CCK-8 Assay, Western Blot, Expressing, Cell Culture, Control

Journal: International journal of molecular medicine

Article Title: Indoleamine 2,3-dioxygenase, by degrading L-tryptophan, enhances carnitine palmitoyltransferase I activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

doi: 10.3892/ijmm.2016.2750

Figure Lengend Snippet: Figure 6. Effect of indoleamine 2,3-dioxygenase (IDO) on forkhead box P3 (FoxP3) and related orphan receptor γt (RORγt) expression in CD4+ T cells derived from mixed lymphocyte reactions (MLRs) with or without oleate. Ten MLRs were performed in culture media containing or not oleate and in the presence or absence of the IDO inhibitor 1MT. CD4+ T cells were then isolated to evaluate the expression of FoxP3 and RORγt. (A) Western blot analysis results of 5 of 10 performed experiments with oleate are shown. (B) IDO increased FoxP3 expression, (C) but decreased RORγt expression in MLR-derived CD4+ T cells. (D) Western blot analysis results of 5 of 10 performed experiments without oleate are shown. In this case, (E) IDO increased FoxP3 expression, (F) whereas it decreased RORγt expression in MLR-derived CD4+ T cells. However, the presence of oleate in the culture medium demonstrated a more profound effect of IDO on FoxP3 expression. Error bars represent the means ± SD; **p<0.001 compared to MLR-devided cells.

Article Snippet: The primary antibodies used in western blot analysis were specific for eIF2α phosphorylated at serine 51 (p-eIF2α; Cat. no. 9721) (Cell Signaling Technology), cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1; Cat. no. sc-20772) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p-70S6 kinase phosphorylated at threonine 389 (p-p70S6K; Cat. no. 9234) (Cell Signaling Technology), CPT1A (Cat. no. 12252S; Cell Signaling Technology), CPT1B (Cat. no. ab134988), CPT1C (Cat. no. ab87498) (both from Abcam), acetyl-CoA carboxylase 2 (ACC2; Cat. no. 8578) (Cell Signaling Technology), ACC2 phosphorylated at serine 221 (p-ACC2; Cat. no. ab109540) (Abcam), activated cleaved at aspartate 175 caspase-3 (Cat. no. 9664), forkhead box P3 (FoxP3; Cat. no. 5298) (both from Cell Signaling Technology), retinoic acid receptor related orphan receptor γt (RORγt; Cat. no. orb6888) (Biorbyt, Cambridge, UK) and β-actin (Cat. no. 4967; Cell Signaling Technology).

Techniques: Expressing, Derivative Assay, Isolation, Western Blot