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: 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