Journal: iScience

Article Title: Identifying G protein-coupled receptors involved in adipose tissue function using the innovative RNA-seq database FATTLAS

doi: 10.1016/j.isci.2023.107841

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Article Snippet: siPtger2, rArGrArUrGrArArArCrArGrArCrUrUrUrArUrGrArGrGrUAG , Origene , SR412228, siRNA C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Amplified Luminescent Proximity Homogenous Assay, Functional Assay, Luminescence Assay, Reverse Transcription, Gene Expression, Plasmid Preparation, Software, Imaging, Cytometry

Journal: Nature neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling.

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: Fig. 7 | PGE2 enhances prion neurotoxicity mainly through the EP4 receptor (Ptger4). a,b, Live-cell imaging (a) and quantitative analysis (b) of chronically prion-infected HovS cells expressing control (Ctrl) transgene or one of the four PGE2 receptors (Ptger1–4). Effects of PGE2 treatment on prion-induced cell toxicity were measured with the ratio of GFP signals under the PGE2 condition against the DMSO condition; n = 4 independent experiments. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons: P < 0.0001 (Ptger1 versus Ctrl); P = 0.2246 (Ptger2 versus Ctrl); P = 0.3351 (Ptger3 versus Ctrl); P < 0.0001 (Ptger4 versus Ctrl). c, Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of prion- infected primary neurons treated with different concentrations of Ptger4 agonist L902688. d, Quantification of neuronal density as well as Map2-positive and Tau positive areas shown in c; n = 6 independent experiments. Data are presented as

Article Snippet: Lenti-vectors used for expressing human PGE2 receptors EP1 (pLenti-PTGER1-mGFP-P2A-Puro, cat. no. RC208597L4), EP2 (pLenti-PTGER2-mGFP-P2A-Puro, cat. no. RC210883L4), EP3 (pLenti-PTGER3-mGFP-P2A-Puro, cat. no. RC220173L4) and EP4 (pLenti-PTGER4-mGFP-P2A-Puro, cat. no. RC210932L4) were purchased from OriGene Technologies.

Techniques: Live Cell Imaging, Infection, Expressing, Control, Immunofluorescence

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 1 Prostaglandin receptor expression in human CD4+ T cell subsets. (A) Quantitative RT-PCR analysis of EP1, EP2, EP3, and EP4 expression in distinct CD4+ T cell subsets. P was calculated by 1-way ANOVA test. n = 10 individual donors. CD4+ subsets were differentiated from naive CD4+ cells in the presence of 2.5 μg/ml anti-CD3 antibodies, 1 μg/ml anti-CD28 antibodies, and the respective cytokines required for the induction of specific CD4+ subsets. Representative example (B) and cumulative results (C) of flow cytometric analysis of EP2 expression in Th0, Th1, Th2, Th9, Th17, and Tregs. P was calculated by 1-way ANOVA test. n = 8 individual donors. (D) IL-17A, RORC, and EP2 expression in ex vivo–isolated IL-17+CD4+ T cells and IL-17–CD4+ T cells. Cells were sorted by FACSAria based on cell surface expression of IL-17. ***P < 0.001. P was calculated by an unpaired Student’s t test; mean ± SEM. n = 10 individual donors. (E) Representative example of flow cytometric analysis of EP2 expression in IFN-γ+ and IL-17+ memory CD4+ T cells. Cells were analyzed for EP2 expression directly after ex vivo isolation without prior purification by FACSAria cell sorting.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Ex Vivo, Isolation, Purification, FACS

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 2 Expression of EP2 negatively correlates with RORC expression in CD4+ T cells. (A) Naive CD45RA+CD45RO–CD4+ T cells from healthy controls were cultured with the indicated cytokines. EP2 and RORC expression was analyzed by RT-PCR. *P < 0.01 by 1-way ANOVA (mean ± SEM; n = 10). (B) Time kinetics of EP2 and RORC expression in Th17 cells induced from naive CD45RA+CD45RO–CD4+ T cells. ***P < 0.0001 by an unpaired Student’s t test comparing EP2 expression at 0 and 24 hours; mean ± SEM. n = 5.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 4 EP2 expression in CD4+ T cells is induced by TCR stimulation. (A) Strong TCR stimulation partly restored EP2 expression in RORC+CD4+ T cells. Naive CD4+ T cells were transfected with a RORC expression plasmid and cultured with various concentrations of anti-CD3 anti- bodies under nonskewing (Th0) conditions. EP2 expression was analyzed and compared with EP2 expression in mock-transfected Th0 cells. P = 0.0006 comparing EP2 expression at 2.5 μg and 0 μg anti-CD3 mAbs, and P = 0.0057 comparing EP2 expression at 10 μg and 0 μg anti-CD3 mAb; mean ± SEM. n = 5. (B) RORC expression levels were controlled in RORC-transfected Th0 cells.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 5 EP2 overexpression induces a pathogenic genotype in Th17 cells. (A) Quantitative RT-PCR analysis of genes in EP2-transfected Th17 cells and mock-transfected Th17 cells (mean ± SEM; n = 5). *P < 0.05. (B) Fold changes in gene expression after treatment of EP2-transfected and mock-transfected Th17 cells with butaprost and 5 μg/ml anti-CD3. (C) Cytokine secretion by EP2-expressing Th17 cells. EP2-overexpressing cells and mock-transduced Th17 cells were activated with an EP2 agonist in the presence of 5 μg/ml anti-CD3. Secretion of IFN-γ, GM-CSF, and IL-10 was analyzed by ELISA (mean ± SEM; n = 5). All P values were calculated by an unpaired Student’s t test.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 6 EP2 expression is increased in Th17 cells from MS patients. (A) EP2 expression in Th17 cells from healthy controls (HC) and Th17 cells from MS patients were compared using RT-PCR (mean ± SEM; n = 20). Representative example (B) and cumulative results (C) of flow cytometric analysis of EP2 in Th17 cells from MS patients and healthy controls. n = 7. Percentage of positive cells and mean fluorescence intensity ratio (MFIR) = MFI (EP2) – MFI (isotype) are shown. Representative example (D) and cumulative results (E) of flow cytometric analysis of IL-17A and IFN-γ in CD4+ T cells from MS patients and healthy controls. CD45RO+ memory CD4+ T cells from healthy controls and MS patients were cultured for 4 days with IL-23. Where indicated, cells were cultured in the presence of PGE2, an EP2 agonist (butaprost), or an EP4 agonist (misoprostol). Percentage of IL-17A+IFN-γ+ Th17 cells in MS patients and healthy controls are shown. *P < 0.01. n = 10.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Cell Culture

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 7 EP2 activation drives cytokine production in Th17 cells from MS patients. (A) RT-PCR analysis of IFNG and CSF2 mRNA expression in Th17 cells from MS patients and healthy controls (mean ± SEM; n = 5). Th17 cells were differentiated for 4 days from naive CD4+ T cells. Where indicated, cells were cultured in the presence of the EP2 agonist butaprost. (B) Secretion of GM-CSF by Th17 cells from MS patients and healthy controls was analyzed by ELISA; mean ± SEM. n = 5. All P values were calculated by an unpaired Student’s t test.

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 8 Influence of TCR signaling strength on EP2 expression and RORC binding to PTGER2 in Th17 cells. (A) Proliferative response of CD4+ T cells from healthy controls and MS patients. Cells were cultured for 3 days in the presence of different anti-CD3 antibody concentrations, and prolif- eration was measured by CFSE dilution using flow cytometry. *P < 0.05. (B) CD4+ T cells from MS patients upregulated EP2 at lower anti-CD3 concentrations as compared with those in healthy controls. *P < 0.05. (C) Murine Th17 cells were stimulated with different concentrations of anti-CD3 antibodies, and binding of RORγt to the promoter region (5′ end) and the 3′ end of Ptger2 was analyzed by quantitative real-time PCR of ChIP-PCR. Dashed line indicates background using cells from RORγt–/– mice. Binding of RORγt to the Il17 locus was used as a control. (D) ChIP-PCR analysis of RORC binding to the PTGER2 promoter region (5′ end) in Th17 cells from MS patients and healthy controls. Th17 cells were stimulated with different concentrations of anti-CD3 antibodies (mean ± SEM; n = 3). (E) TCR signaling–dependent EP2 expression in Th17 cells from MS patients and healthy controls was analyzed by RT-PCR (mean ± SEM; n = 3).

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Cell Culture, Flow Cytometry, Real-time Polymerase Chain Reaction, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Clinical Investigation

Article Title: Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells

doi: 10.1172/jci72973

Figure Lengend Snippet: Figure 9 Model for PGE2 effects on Th17 cells in healthy individuals and autoimmune disease. TCR-induced EP2 upregulation is inhibited by RORC in Th17 cells from healthy individuals. In Th17 cells from patients with autoimmune disease, RORC-mediated PTGER2 silencing is diminished and EP2 is expressed. EP2 signaling in pathogenic Th17 cells promotes the induction of IL-17A+IFN-γ+ cells and induces a potentially pathogenic Th17 cell phenotype (26).

Article Snippet: For gene overexpression, an EP2 gene expression construct (OriGene Technologies) or a mock control construct was delivered into Th17 cells by electroporation nucleofection (Amaxa) according to the manufacturer’s instructions.

Techniques: