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: Frontiers in Immunology

Article Title: Macrophage TRIM21 lactylation exacerbates infection-induced orchitis through enhancing STAT1-mediated CXCL9 and CXCL10 production

doi: 10.3389/fimmu.2025.1684836

Figure Lengend Snippet: CXCL9 and CXCL10 contribute to inflammatory cell infiltration and impair spermatogenesis during infection-induced orchitis. (A) Venn diagram showing that chemokines CXCL9, CXCL10, and CXCL11 exhibit chemotactic effect on monocytes/macrophages, T cells, and dendritic cells, while CCL17 and CCL22 only target T cells. (B) Relative mRNA levels of Cxcl9 , Cxcl10 , Cxcl11 , Ccl17 , and Ccl22 in the testes from LPS-induced orchitic and PBS control mice ( n = 3). (C) Relative mRNA levels of Cxcl9 , Cxcl10 , Cxcl11 , Ccl17 , and Ccl22 in the testes from UPEC-induced orchitic and vehicle control mice ( n = 3). (D) Gross morphology of the testes from LPS-induced orchitic mice, orchitic mice with isotype IgG injection, and orchitic mice with single or dual neutralization of CXCL9 and CXCL10. Scale bar, 1 cm. (E) Weight and relative weight of the testes from the sham group of mice and aforementioned mice in (B) ( n = 3). (F) Comparison of representative seminiferous tubules’ diameter and seminiferous epithelium thickness between the sham group and the above groups ( n = 3). (G) Quantified flow cytometry showing the number of macrophages (CD45 + CD11b + F4/80 + ) per 10 6 testicular cells in the sham group and the above groups ( n = 3–4). (H) Representative H&E staining images of seminiferous tubules, testicular interstitium, caput epididymis, and cauda epididymis from the indicated mice mentioned above. In the testicular interstitium, different arrows indicate different immune cells as shown. We rely on the characteristic nuclear morphology and localization of immune cells as a supportive, histological complement to the flow cytometric data in (G) . Scale bars, 100 μm at 20× magnification and 20 μm at 100× magnification. Two groups of data are statistically compared with Student’s t- test, while multiple groups of data are statistically compared with one-way ANOVA. Error bars represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: The recombinant neutralizing antibodies anti-CXCL9 (AF-492-NA, R&D Systems, Minneapolis, MN, USA) and anti-CXCL10 (AF-466-NA, R&D Systems, Minneapolis, MN, USA) were diluted in sterile PBS and stored at −20°C.

Techniques: Infection, Control, Injection, Neutralization, Comparison, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Macrophage TRIM21 lactylation exacerbates infection-induced orchitis through enhancing STAT1-mediated CXCL9 and CXCL10 production

doi: 10.3389/fimmu.2025.1684836

Figure Lengend Snippet: Macrophages are a predominant cellular source of CXCL9 and CXCL10 in the testis during LPS-induced orchitis. (A) Flow cytometry showing immune cells (gated on CD45 + ) expressing chemokines CXCL9 or CXCL10 in the testes from LPS-induced orchitic mice, with the proportion of macrophages identified. (B) Flow cytometry showing the proportion of testicular macrophages 3 days after in situ injection of clodronate liposomes in the testes. PBS liposomes were injected as control, while the sham group served as the negative control. (C) Quantified flow cytometry showing the number of macrophages (CD45 + CD11b + F4/80 + ) per 10 6 testicular cells in LPS-induced orchitic mice and sham/orchitic mice with in situ injection of clodronate liposomes/PBS liposomes. The sham group of mice served as the negative control. Sample sizes: sham = 7; LPS + PBS = 6; clodronate liposome + PBS = 4; PBS liposome + PBS = 4; clodronate liposome + LPS = 6; PBS liposome + LPS = 6. (D) Representative immunofluorescence images of the testes showing the co-localization of F4/80 + macrophages and chemokines CXCL9 or CXCL10 in mice with PBS/LPS/clodronate liposomes/PBS liposomes + LPS/clodronate liposomes + LPS intratesticular injection. Scale bar, 100 μm at 20× magnification. (E) Representative H&E staining images of seminiferous tubules, caput epididymis, and cauda epididymis from the indicated mice mentioned in (D) . Scale bar, 100 μm at 20× magnification. Data are statistically compared with one-way ANOVA. Error bars represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: The recombinant neutralizing antibodies anti-CXCL9 (AF-492-NA, R&D Systems, Minneapolis, MN, USA) and anti-CXCL10 (AF-466-NA, R&D Systems, Minneapolis, MN, USA) were diluted in sterile PBS and stored at −20°C.

Techniques: Flow Cytometry, Expressing, In Situ, Injection, Liposomes, Control, Negative Control, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Macrophage TRIM21 lactylation exacerbates infection-induced orchitis through enhancing STAT1-mediated CXCL9 and CXCL10 production

doi: 10.3389/fimmu.2025.1684836

Figure Lengend Snippet: Lactylation promotes the transcriptional expression of CXCL9 and CXCL10 in macrophages. (A) The lactate levels in the testes from LPS-induced orchitic mice, PBS control, and sham group ( n = 6). (B) Representative immunofluorescence images showing the general localization of lysine lactylation modification (pan-Kla) and F4/80 + macrophages in the testes from mice mentioned in (A) . Scale bar, 200 μm at 10× magnification. (C) More precise co-localization of pan-Kla-positive signals and F4/80 + macrophages in the testicular interstitium under an oil immersion microscope. White dotted lines indicate the edge of the seminiferous tubules, and three yellow boxes indicate typical F4/80 + macrophages in the testicular interstitium. Scale bar, 20 μm at 100× magnification. (D) Co-localization analysis of pan-Kla and F4/80 fluorescent signals in the testes from LPS-induced orchitic mice, PBS control, or sham group. (E) Intracellular lactate levels of RAW264.7 cells 24 h after LPS + IFN-γ stimulation with/without oxamate (Oxa) or lactate (Lac) treatment, or after incubating with Oxa or Lac alone. RAW264.7 cells without LPS + IFN-γ stimulation served as control (Ctl). Pound signs (#) indicate comparison with the LPS + IFN-γ stimulation group ( n = 5, normalized with intracellular protein levels). (F) Global lysine lactylation levels in RAW264.7 cells from the groups mentioned in (E) . (G) Relative mRNA levels of Cxcl9 and Cxcl10 in RAW264.7 cells from the groups mentioned in (E) ( n = 3). Pound signs (#) indicate comparison with the LPS + IFN-γ stimulation group. Data are statistically compared with one-way ANOVA. Error bars represent mean ± SEM. * p < 0.05, ## p < 0.01, ### p < 0.001; ns, not significant.

Article Snippet: The recombinant neutralizing antibodies anti-CXCL9 (AF-492-NA, R&D Systems, Minneapolis, MN, USA) and anti-CXCL10 (AF-466-NA, R&D Systems, Minneapolis, MN, USA) were diluted in sterile PBS and stored at −20°C.

Techniques: Expressing, Control, Immunofluorescence, Modification, Microscopy, Comparison