cxcl9  (R&D Systems)

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: A total of 10 × 10 4 cells/well were suspended in RPMI 1640 containing 0.2% FBS in the upper chamber, and the lower chamber was filled with the conditioned medium of BMDMs from C57BL/6 mice with or without recombinant mouse CXCL9 protein (492-MM-010/CF, R&D Systems).

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

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 influences the activation of the STAT1-CXCL9 signaling pathway. A , B CXCL9 mRNA levels in THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) as determined by RT–qPCR. β-actin was used as an internal control (representing 2 independent experiments). C Detection of CXCL9 mRNA levels in BMDMs isolated from WT and Ccng2 −/− C57BL/6 mice by RT-qPCR. GAPDH was used as an internal control (representing 2 independent experiments). D The STAT1 and p-STAT1 (Y701) protein levels were determined in the THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2) by western blotting. β-tubulin was used as a loading control. E The STAT1 and p-STAT1 (Y701) protein levels were determined in the THP-1 stable cell lines (Vector and Flag-cyclin G2) by western blotting. β-tubulin was used as a loading control (representing 3 independent experiments). F STAT1 protein levels in the cytoplasm and nucleus of THP-1 cells were detected by western blotting (Nonsense and shcyclin G2#1). β-tubulin was used as a cytoplasmic loading control. Lamin B1 was used as a nuclear loading control. G STAT1 immunofluorescence staining for THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2), representative images are shown. Scale bar = 10 μm. ( A – C ) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD.**** p < 0.0001

Article Snippet: A total of 10 × 10 4 cells/well were suspended in RPMI 1640 containing 0.2% FBS in the upper chamber, and the lower chamber was filled with the conditioned medium of BMDMs from C57BL/6 mice with or without recombinant mouse CXCL9 protein (492-MM-010/CF, R&D Systems).

Techniques: Activation Assay, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Control, Isolation, Western Blot, Immunofluorescence, Staining

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 knockout in macrophages attenuates the inhibitory effects of IFN-γ on colon cancer cell growth. A–C MC38 cells were mixed with BMDMs from WT and Ccng2 −/− C57BL/6 mice at a ratio of 5:1 and injected subcutaneously into C57BL/6 mice, which were then treated with IFN-γ at specific times. Gross tumors ( A ), tumor weights ( B ), and tumor volumes ( C ) were measured at the endpoint. Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SEM ( n = 5). D A schematic model depicting the role of cyclin G2 in macrophages after IFN-γ treatment. Upregulated cyclin G2 after IFN-γ treatment inhibited the interaction between PP2Ac and STAT1, thereby increasing the nuclear import of STAT1 and promoting CXCL9 transcription. Increased CXCL9 secretion can promote CTL chemotaxis and inhibit vascular endothelial cell angiogenesis, ultimately inhibiting tumor progression. ** p < 0.01; **** p < 0.0001

Article Snippet: A total of 10 × 10 4 cells/well were suspended in RPMI 1640 containing 0.2% FBS in the upper chamber, and the lower chamber was filled with the conditioned medium of BMDMs from C57BL/6 mice with or without recombinant mouse CXCL9 protein (492-MM-010/CF, R&D Systems).

Techniques: Knock-Out, Injection, Chemotaxis Assay