mouse cxcl9 dy492  (R&D Systems)

Journal: bioRxiv

Article Title: Selective JAK Inhibition Reveals Paradoxical and Hierarchical Control of interferon-γ-driven Autoimmunity in AIRE Deficiency

doi: 10.64898/2026.03.05.709894

Figure Lengend Snippet: Untreated Air e −/− mice and Air e −/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Lungs were processed for intracellular cytokine staining, qPCR, ELISA, and immunoblot analyses. ( A-B ) Representative flow cytometry plots showing IFN-γ production by CD4 + and CD8 + T cells. ( C-D ) Frequency (of total CD4 + and CD8 + T cells) and absolute numbers of IFN-γ + CD4 + and IFN-γ + CD8 + T cells in the lung. ( E-F ) Relative Ifng mRNA expression and IFN-γ protein concentrations in lung homogenates. ( G ) Relative Stat1 mRNA expression. ( H ) Representative immunoblots of phospho-STAT1 (pSTAT1), total STAT1, and GAPDH. ( I-J ) Quantification of total STAT1 and phospho-STAT1 normalized to GAPDH. For IFN- γ + CD4 + and IFN-γ + CD8 + T cells: n = 9-14 mice per group from four independent experiments. For Ifng and Cxcl9 mRNA: n = 15-22 mice per group from four independent experiments. For Stat1 _mRNA: n = 10-17 mice per group from three independent experiments. For CXCL9 protein: n = 5-10 mice per group from two independent experiments. For STAT1 and pSTAT1 immunoblots: n = 15-22 mice per group from four independent experiments). Statistical analyses were performed using one-way ANOVA or Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Air e −/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: A DuoSet® ELISA kit (catalog no. DY492-05; R&D Systems, USA) was used to measure CXCL9 and a high-sensitivity ELISA kit was used for IFN-γ (catalog no. 88-8314-88; Invitrogen, USA).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: Loss of miR-29a/b1 cluster reprograms the tumor microenvironment and contributes to immunosuppression in lung cancer

doi: 10.1158/2326-6066.CIR-25-1060

Figure Lengend Snippet: A. The PD1R1 control (ctrl) and miR-29a/b1 re-expressing tumors from Figure 4C were analyzed for Cd8a expression by qPCR. Rpl32 was used as an internal control. This qPCR was completed one time with 3 independent tumors. *p<0.05 by Welch’s t test. B. CD8 infiltration into PD1R1 control and miR-29 tumors was assayed by IHC staining. Staining was completed once on 3 mice/group, with 5 independent regions of the tumor imaged and CD8 signal quantified using ImageJ software. The CD8 signal per field of view (FOV) is depicted to the right. n = 3 mice/group, 5 ROIs/tumor. Scale bar = 100 μm. Zoom on inset = 2.5x. *p<0.05 by Welch’s t test. C. Tumors from B were also stained by IHC for Granzyme B. Representative images are shown. Scale bar = 100 μm. Zoom on inset = 2.0x. p = 0.0571 by Welch’s t test. D. A representative tumor lysate from the PD1R1-miR-29a/b1 and control tumors were processed via a Proteome Profiler that includes 111 various chemokines/cytokines. Colored boxes indicate those factors which were differentially expressed between the control and miR-29 tumors, with matching colored text to the right to indicate the specific markers. This was completed once. E. The analytes highlighted on the cytokine array in panel D were assayed via specific ELISAs in the PD1R1-miR-29a/b1 and control tumor replicates. p values are indicated as measured by unpaired t test. ELISAs were completed twice for CXCL9 and CXCL10, and once for the other hits. F. PD1R1-miR-29a/b1 and control tumor cell lines were treated with doxycycline for 24 hours to induce miR-29a/b1 expression, and cells were then collected for RNA. qPCR was performed to determine transcript levels of analytes identified from the array in D, and Rpl32 was used as an endogenous control. This was completed twice. *p<0.05 by unpaired t test.

Article Snippet: To follow up on potential hits from the cytokine array on multiple tumors per group, we utilized commercially available ELISAs specific for mouse CXCL9, CXCL10, CXCL11, CD93, IL1ra, CD105 (Catalog numbers in order: DY492, DY466, DY572, MCD930, MRA00, MNDG00; R&D Systems).

Techniques: Expressing, Control, Immunohistochemistry, Staining, Software

Journal: bioRxiv

Article Title: Histone methyl-transferase G9a inhibition boosts the efficacy of immune checkpoint inhibitors in experimental hepatocellular carcinoma

doi: 10.1101/2025.07.08.663670

Figure Lengend Snippet: (A) Effect of CM272 and EZM8266 on IFNγ-triggered CXCL10 production in murine (PM299L) and human (HuH7) HCC cells. Cells were treated with CM272 for 48 h or with CM272 for 24 h and then with INFγ (75 U/mL) for another 24 h, or with INFγ alone for 24 h. PM299L were treated with 400 nM and HuH7 received 1 µM of CM272. For EZM8266, cells were pretreated for 48 h with EZM8266 (5 µM) and then with IFNγ (75 U/mL) for another 24 h as indicated. CXCL10 protein levels were measured by ELISA in cells’ conditioned media. (B) Effect of G9a inhibition with CM272 or EZM8266 on the expression of MHC class I complex protein (MHC-I) on the surface of PM299L cells. Cells were treated with IFNγ and CM272 or EZM8266 as indicated in panel A, and MHC-I levels were determined by FACS analysis. (C) ChIP analyses of H3K9me2 levels in the proximal promoter regions of CXCL10 and HLA-A genes in HuH7 cells treated with IFNγ (75 U/mL) and CM272 (400 nM) as indicated in panel A. (D) Evaluation of the expression of Transposable elements (TEs) and endogenous retroviral sequences (ERVs) by RNA-seq in NM53 murine HCC cells treated with IFNγ, CM272 and their combination as indicated in panel A. (E) Immunofluorescence analyses of dsRNA in PM299L HCC cells treated with CM272 (24 h) or EZM8266 for (48 h) h. Right panel shows a control without primary antibody. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: CXCL9 and CXCL10 concentrations were measured in culture supernatants collected at the end of the incubation periods using commercial ELISA kits for mouse (CXCL9: R&D Systems, DY492; CXCL10: R&D Systems, DY466) and human cell lines (CXCL10: BD Biosciences, 550926).

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Retroviral, RNA Sequencing, Immunofluorescence, Control

Journal: bioRxiv

Article Title: TBK1 and IKKε protect target cells from IFNγ-mediated T cell killing via an inflammatory apoptotic mechanism

doi: 10.1101/2024.08.06.606693

Figure Lengend Snippet: (A) Global transcriptomics analysis of enriched gene pathways measured as log2 fold change between IFNγ - stimulated vs unstimulated control (Ctrl) WT cells (left), IFNγ - stimulated vs unstimulated control (Ctrl) DKO cells (middle), and a ratio of the IFNγ-stimulated change in DKO versus IFNγ-stimulated change in WT cells (right). (B) Heatmap expression profile of inflammatory genes obtained from RNAseq analysis of WT and DKO B16 cells stimulated with IFNγ (100 ng/mL) for 0, 16, 24 h. (C) Global proteomics analysis of enriched proteins in pathways measured as log2 fold change between IFNγ - stimulated vs unstimulated control (Ctrl) WT cells (left), IFNγ - stimulated vs unstimulated control (Ctrl) DKO cells (middle), and a ratio of the IFNγ-stimulated change in DKO versus IFNγ-stimulated change in WT cells (right). (D) Western blot analysis of the indicated proteins in nuclear extracts obtained from WT, DKO, TNFR1 TKO, ZBP1 TKO, and QKO B16 cells treated with IFNγ (100 ng/mL) for 0 or 24 h. (E) ELISA analysis of mouse CCL2 and CXCL9 in WT, DKO, TNFR1 TKO, ZBP1 TKO, and QKO B16 cells treated with IFNγ (100 ng/mL) for 0 or 24 h. ns = not significant, **P<0.01, ****P<0.0001, statistical analysis was performed using unpaired t test. (F) Western blot analysis of the indicated proteins in WT and DKO B16 cells treated with TNF (10 ng/mL) for 0, 15, 30, and 60 min.

Article Snippet: The ELISA kits were obtained from the following sources: mouse TNFα (BioLegend, 430904), mouse IFNγ (BioLegend, 430801), mouse MCP-1 (CCL2) (BioLegend, 432704), and mouse MIG (CXCL9) (Bio-Techne, DY492-05).

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay