Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 can all bind and signal through the chemokine receptor CXCR3 that is typically found on T cells. (B) The EMBL-ELI expression atlas (human) was analysed for relatedness in expression of CXCR3 and its’ ligands CXCL9, 10 and 11 across all tissues or (C) in distinct tissues and diseases.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of the in vivo air pouch leukocyte recruitment model. (B) Analysis of chemokine concentration in the carrageenan inflamed air pouch. (C) Representative tSNE of all murine cells gated on live, single, CD45 + and built on CD4, CD8, F4/80, Ly6C, Ter119, CD3, TCRβ, CXCR3, Ly6G, CD11c, B220, CD11b, CD64, Siglec F, NK1.1 and TCRγδ. FlowSOM clusters are illustrated by gates. (D) tSNE analysis of air pouches injected with equimolar amounts of CXCL9, 10 and 11. (E) Quantification of all leukocytes (CD45 + ) and T cells within the air pouch following injection of CXCL9, 10 or 11. E analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: In Vivo, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of HS GAG structure, including sulphation points and the enzymes that produce them. (B) Normalised (relative to wild type) binding of labelled CXCL9, 10 or 11 to genetically modified CHO cells. (C) Normalised and absolute binding of CXCL9, 10 and 11 to CHO cells in which KS^ST1/2/3 have been genetically removed. (D and E) Normalised binding of CXCL9, 10 and 11 to CHO cells genetically engineered to express the enzymes regulating 3-O GAG sulphation. (F) EMBL-ELI expression atlas analysis of relatednessCXCL9, 10 and 11 and GAG sulphation gene expression. B and D, data plotted as mean from three separate pooled experiments. C and E data plotted as mean ± SEM from three separate pooled experiments and analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Binding Assay, Genetically Modified, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 all bind to the same receptor with different affinities and biased signalling outcomes and are found in over-lapping expression patterns during inflammation and disease. (B) Differential GAG interactions means that CXCL9 is more likely to be retained on GAGs on the cell surface or within the ECM, with CXCL10 and CXCL11 being more likely to be present in their soluble state.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: EBioMedicine

Article Title: Histone deacetylases inhibitor chidamide synergizes with humanized PD1 antibody to enhance T-cell chemokine expression and augment Ifn-γ response in NK-T cell lymphoma.

doi: 10.1016/j.ebiom.2022.104420

Figure Lengend Snippet: Fig. 5: Anti-PD1/chidamide shows an immune response with epigenetic regulation. a, Western blot of Ifn-γ, Cxcl9, Cxcr3, Acetylated histone H3, histone H3, Pd-l1, and β-actin in anti-PD1/chidamide and each monotherapy. b, Western blot of Ifn-γ, Cxcl9, Cxcr3, Acetylated histone H3,

Article Snippet: Tissue homogenates were detected in the Cxcl9 ELISA kit (CSB-EL006252MO, Cusabio) and Ifn-γ ELISA kit (KE10001, Proteintech).

Techniques: Western Blot

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 2. RGS1 is a modulator of IFNγ-JAK-STAT1 signaling. a Differentially expressed cancer-related gene sets (H_Hallmarks) with high RGS1 expression in the KIRC cohort, LUAD cohort, LUSC cohort, and SKCM cohort from TCGA. NES, normalized enrichment score. FDR q, false discovery rate q value. b – d GSEA output of genes in the HALLMARK_INTERFERON_GAMMA_RESPONSE by RGS1 high and low expression groups from the KIRC cohort (b), LUAD cohort (c) and LUSC cohort (d) in the TCGA database. ES, enrichment score. e – l Analysis of IFNγ-STAT1 signaling in 786O and Renca cells. Cell lysates of 786O and Renca cells stably expressing nc or shRGS1 (e, g) and Vector or RGS1-OE (i, k) were analyzed by western blotting using RGS1, STAT1, P-STAT1 (Y701), IFNGR1, and IRF1 antibodies. Tublin was used as an internal control. f, j IFNγ-inducible gene expression in 786O cells. mRNA expression of IRF1, IRF9, STAT1, and IFNGR1 were detected by real-time qPCR. Actin was used as an internal control. h, l IFNγ-inducible gene expression in Renca cells. mRNA expression of Irf1, Irf9, and Stat1 were detected by real-time qPCR. Actin was used as an internal control. Cells in e, f, i, j were stimulated with 10 ng/ml human recombinant IFNγ or 0.1% BSA negative control for 2 h. Cells in g, h, k, l were stimulated with 5 ng/ml mouse recombinant IFNγ or 0.1% BSA negative control for 2 h. m, n IFNγ-induced CXCL9 secretion. Renca (m) and LLC (n) cells were cultured in serum-free medium and treated with 5 ng/ml IFNγ for 24 h. The concentration of CXCL9 was analyzed using an ELISA kit. o, p Cell surface levels of IFNGR1 in nc or shRGS1 (o) and Vector or RGS1-OE (p) 786O cells (pre-gated with FSC-A vs. SSC-A, and FSC-A vs. FSC-H). Cells were treated with 10 ng/ml IFNγ for 2 h. Right, quantification of the mean fluorescence intensity (MFI). Unpaired t-test was performed with GraphPad Prism 9. All data are representative of three independent experiments. Data in the bar graphs represent mean ± S.D., n = 3. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Control, Gene Expression, Recombinant, Negative Control, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 5. RGS1 is associated with T cell infiltration in RCC and NSCLC mouse models. a, d Representative images and quantification (right) of immunohistochemistry staining of IFNGR1, ATF3 and CXCL9 expression in harvested CTRL and ShRgs1 Renca (a) or LLC (d) subcutaneous tumor sections. The percentages of positively stained area (right) were analyzed using Image J software. Scale bar, 100 µm. b, e T cell infiltration and quantification (below) of Renca (b) or LLC (e) subcutaneous tumor. Paraffin-embedded tissue sections of murine tumors were immunohistochemically stained with antibodies against CD3, CD4, CD8 and PD1. The percentages of positively stained cells (below) were analyzed using Image J software. Scale bar, 100 µm. c, f Representative immunofluorescence images of CD8 and PD1 staining in ShRgs1 or CTRL Renca (c) or LLC (f) tumor sections. White arrows indicate merge of CD8 and PD1 fluorescence signals. Scale bar, 50 µm. Data in the graphs represent mean ± S.D, n = 5. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Immunohistochemistry, Staining, Expressing, Software, Immunofluorescence, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Cyclin G2 in macrophages regulates CTL chemotaxis and vascular endothelial cell tube formation via CXCL9. A CXCL9 levels in the supernatants of BMDMs from WT and Ccng2 −/− C57BL/6 mice treated with IFN-γ were determined by ELISA (representing 3 independent experiments). B , C CXCL9 levels in the supernatants of THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) treated with IFN-γ were determined by ELISA (representing 3 independent experiments). D , E CTL chemotaxis analyzed by treating conditioned medium from BMDMs isolated from WT and Ccng2 −/− C57BL/6 mice treated with or without recombinant CXCL9. Scale bar = 200 μm (representing 3 independent experiments). F Tube formation experiments showed the tube formation ability of SVEC4–10 cells treated with conditioned medium from BMDMs isolated from Ccng2 −/− C57BL/6 mice. The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 500 μm (representing 3 independent experiments). G Tube formation experiments showed the tube formation ability of HUVECs treated with conditioned medium from a THP-1 stable cell line (shcyclin G2#1). The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 200 μm (representing 3 independent experiments). (A–C, E–G) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD ** p < 0.01; *** p < 0.001; ns , not significant

Article Snippet: Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Plasmid Preparation, Isolation, Recombinant

Journal: Annals of Translational Medicine

Article Title: Antibody microarray analysis of serum inflammatory cytokines in patients with calcific aortic valve disease

doi: 10.21037/atm-20-4463

Figure Lengend Snippet: Primers sequences for RT-PCR

Article Snippet: Then protein samples (20–100 µg) were resolved using SDS-PAGE and then moved to polyvinylidene fluoride (PVDF) membrane (Millipore, USA) with being probed with GAPDH (Proteintech), CXCL13/BLC (RRIDs: AB_1191734), IL-12p40/IL12B (RRIDs: AB_1156284), CXCL9/MIG (RRIDs: AB_2086730) followed by appropriate secondary antibody.

Techniques: Sequencing

Journal: Cell death & disease

Article Title: MicroRNA-17-92 cluster promotes the proliferation and the chemokine production of keratinocytes: implication for the pathogenesis of psoriasis.

doi: 10.1038/s41419-018-0621-y

Figure Lengend Snippet: Fig. 3 MiR-17-92 cluster promotes the production of chemokines in keratinocytes primed by cytokines. a The mRNA levels of CCL20, CCL27, CXCL9, CXCL10, CXCL11, and CX3CL1 in NHKs with different treatments and transfections as indicated were analyzed using qRT-PCR. Mean ± SD is shown. Data are representative of three independently performed experiments. *P < 0.05; **P < 0.01; ns not significant. b The culture mediums of NHKs with different treatments and transfections as indicated were analyzed by ELISA to determine the secretion levels of CXCL9 and CXCL10. Mean ± SD is shown. Data are representative of three independently performed experiments. *P < 0.05; **P < 0.01; ***P < 0.001. c The migrations of CD3+

Article Snippet: ELISA analysis on the culture medium of NHKs with indicated treatments or transfections for 48 h was performed using Human CXCL9 ELISA Kit (R&D system, Minneapolis, MN, USA) and Human CXCL10 ELISA Kit (R&D system) according to the manufacturer’s instructions, respectively.

Techniques: Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: MicroRNA-17-92 cluster promotes the proliferation and the chemokine production of keratinocytes: implication for the pathogenesis of psoriasis.

doi: 10.1038/s41419-018-0621-y

Figure Lengend Snippet: Fig. 4 MiR-17-92 cluster promotes the production of chemokines in keratinocytes via suppressing SOCS1. a–c The levels of SOCS1, STAT1, p- STAT1, and IRF-1 in NHKs with different transfections and treatments as indicated were detected by western blotting. β-actin was detected as loading control. Data are representative of three independently performed experiments. d The mRNA levels of CXCL9 and CXCL10 in NHKs with different treatments and transfections as indicated were analyzed using qRT-PCR. Mean ± SD is shown. Data are representative of three individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. e The culture mediums of NHKs with different treatments and transfections as indicated were analyzed by ELISA to determine the secretion levels of CXCL9 and CXCL10. Mean ± SD is shown. Data are representative of three independently performed experiments. **P < 0.01; ***P < 0.001; ns not significant. f The migrations of CD3+ T cells in response to the culture mediums from NHKs with different treatments and transfections as indicated were evaluated using transwell assay. Mean ± SD is shown. Data are representative of three independently performed experiments. ***P < 0.001; ns not significant

Article Snippet: ELISA analysis on the culture medium of NHKs with indicated treatments or transfections for 48 h was performed using Human CXCL9 ELISA Kit (R&D system, Minneapolis, MN, USA) and Human CXCL10 ELISA Kit (R&D system) according to the manufacturer’s instructions, respectively.

Techniques: Transfection, Western Blot, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transwell Assay