sting agonist dmxaa  (InvivoGen)

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 promotes intestinal inflammation resolution dependent on cGAS-STING pathway in the repair model. Mice were treated with 2.5 % DSS for 7 days, and then DSS was withdrawn and replaced with sterile water and intraperitoneally injected with DEL-1-Fc (1 ug/mouse) for 6 days (n = 4–6) (A-E). (A, B) Flow cytometry analysis of CD11b + Ly-6G + neutrophils and their percentage in colonic tissues. (C) Assessment of F4/80 (green) and ARG1 (red) expression and colocalization in the colon tissues of the repair model by immunofluorescence. (D, E) Western blot of iNOS, CD206, and ARG1 in colonic tissues, and densitometric analysis quantified the intensity ratio of the target protein to GAPDH. (F-M) Mice were treated with 2.5 % DSS for 7 days, and then DSS was withdrawn and replaced with sterile water and intraperitoneally injected with DEL-1-Fc (1 ug/mouse) and DMXAA (70 ug/mouse) for 6 days (n = 10). (F) Diagram of the modeling and treatment strategy for the induced repair model. (G) Survival rates. (H) Representative images of colons. (I) Colonic length. (J) Representative images of H&E staining. (K) Histological score. (L) RT-qPCR of cytokines ( Il1β , Il6 , Tnfα , Il10 , Arg1 , Ifnα , and Ifnβ ), normalized to β-actin . (N, M) Western blot of iNOS, CD206, and ARG1 in colonic tissues, and densitometric analysis quantified the intensity ratio of the target protein to GAPDH. Statistical analysis was calculated by one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: DSS was then withdrawn and replaced with sterile water, and simultaneously, the mice were intraperitoneally injected with DEL-1-Fc (1 ug/mouse), or Fc (1 ug/mouse), or the STING pathway agonist DMXAA (70 ug/mouse, Topscience) for 6 consecutive days during the recovery phase.

Techniques: Sterility, Injection, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Staining, Quantitative RT-PCR

Journal: Journal of Advanced Research

Article Title: Developmentally endothelial locus-1 facilitates intestinal inflammation resolution by suppressing the Cmpk2-cGAS-STING pathway and promoting reparatory macrophage transition

doi: 10.1016/j.jare.2025.04.030

Figure Lengend Snippet: DEL-1 induces the ubiquitin – proteasome-dependent degradation of transcription factor Spi1, and further inhibits the Cmpk2-cGAS-STING pathway in macrophages. (A-C) Cmpk2 mRNA (A) and protein (B, C) expression in RAW264.7 macrophages with transfection Cmpk2 overexpression plasmid was determined using RT-qPCR and western blot (n = 4). (D-F) RAW264.7 macrophages were transfected with DEL-1, Spi1, and Cmpk2 overexpression plasmids and corresponding controls for 24–36 h. Cells were pulsed with LPS (1 μg/ml) and the STING pathway agonist DMXAA (1 μg/ml) for 4 h in the acute phase, or withdrawn LPS stimulation and treated with DMXAA for 24 h in the recovery phase (n = 4). The expression of CMPK2 and cGAS-STING pathway related protein were measured by western blot, and densitometric analysis quantified the intensity ratio of the target protein to relevant controls: CMPK2/GAPDH, CGAS/GAPDH, p-STING/STING, p-TBK1/TBK1, and p-IRF3/IRF3. (G) The mRNA expression of Spi1 were determined by RT-qPCR in DEL-1 overexpressed macrophages pulsed with LPS and 24 h post LPS withdrawal. (H, I) DEL-1 overexpressed macrophages treated with cycloheximide (CHX, 60 μg/ml), MG132 (20 uM), and chloroquine (50 uM) in the acute and repair model, and the expression of SPI1 was measured using western blot. Statistical analysis was calculated by student’s t tests or one-way-analysis of variance (ANOVA). ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: DSS was then withdrawn and replaced with sterile water, and simultaneously, the mice were intraperitoneally injected with DEL-1-Fc (1 ug/mouse), or Fc (1 ug/mouse), or the STING pathway agonist DMXAA (70 ug/mouse, Topscience) for 6 consecutive days during the recovery phase.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Sustained STING-IRF7 signaling aggravates LPS-induced endometrial inflammation via excessive neutrophil extracellular traps generation

doi: 10.3389/fimmu.2025.1671848

Figure Lengend Snippet: ICAM-1 was regulated by STING-IRF7-LCN2 axis in endometrium. (A) Heatmap of the first 10 DEGs from STING transcriptome. (B) Quantitative mRNA expression of lcn2 in LPS stimulated Tmem173 gt mice (n=6). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (C) Representative immunoblots of LCN2 in LPS-stimulated STING-deficient mice ( Tmem173 gt ) endometrium tissues, compared with WT-LPS stimulated mice. (n=5). (D) Quantification of the amount of LCN2 in the four groups (n=4). One-way ANOVA test was applied with **** P <0.0001. (E) Representative immunoblots of LCN2 in EECs transfected with IRF7 for 48 (h) (F) Quantification of the amount of LCN2 in the two groups (n=4). Student’s t test was applied with *** P <0.001. (G) Quantitative mRNA expression of mc4r in LPS stimulated Tmem173 gt mice (n=6). One-way ANOVA test was applied with *** P <0.001 (WT vs. Tmem173 gt in LPS group). (H) Representative immunoblots of MC4R in LPS-stimulated STING-deficient mice ( Tmem173 gt ) endometrium tissues, compared with WT-LPS stimulated mice. (n=5). (I) Quantification of the amount of MC4R in the four groups (n=4). One-way ANOVA test was applied with **** P <0.0001. (J) Representative immunohistochemical staining of MC4R in endometrial tissues of WT or STING-deficient mice ( Tmem173 gt ) stimulated with LPS for 24 h at 400 × magnification (scale bar = 25 μm). (K) The area of MC4R were quantified (n=6). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (L) Quantitative mRNA expression of mc4r in LPS stimulated Lcn2 -/- mice (n=6). Student’s t test was applied with ** P <0.01. (M) Quantitative mRNA expression of mc4r in DMXAA stimulated Lcn2 -/- mice (n=6). Student’s t test was applied with **** P <0.0001. (N) Representative immunoblots of MC4R and ICAM-1 in DMXAA stimulated Lcn2 -/- mice endometrium tissues, compared with WT-LPS stimulated mice. (n=5). (O) Representative immunohistochemical staining of MC4R in endometrial tissues of WT or Lcn2 -/- mice stimulated with LPS for 24 h at 400 × magnification (scale bar = 25 μm). (P) Representative immunoblots of MPO and ICAM-1 in endometrial tissues of WT or Lcn2 -/- mice treated with LPS for 24 (h) (Q) Representative immunohistochemical staining of ICAM-1 in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). (R) Representative immunoblots of ICAM-1 in EECs transfection with si-IRF7 (1,2,3). (S) Quantification of the amount of ICAM-1 in the four groups (n=4). One-way ANOVA test was applied with * P <0.05. (T) Representative immunoblots of citH3 in endometrial tissues of WT or Lcn2 -/- mice treated with LPS for 24 (h) (U) Representative immunoblots of citH3 and ICAM-1 in EECs-neutrophils coculture system with the stimulation of ZINC00640089. (V) Quantification of the amount of ICAM-1 in the two groups (n=5) (ctrl vs. ZINC00640089). Student’s t test was applied with ** P <0.01. (W) Quantification of the amount of citH3 in the four groups (n=5) (ctrl vs. ZINC00640089). Student’s t test was applied with * P <0.05.

Article Snippet: STING agonist DMXAA (HY-10964, MedChemExpress, USA) were diluted in 5% DMSO and 95% saline.

Techniques: Expressing, Western Blot, Transfection, Immunohistochemical staining, Staining, Control

Journal: PLOS Pathogens

Article Title: Antiviral and immune modulatory activities of STING agonists in a mouse model of persistent hepatitis B virus infection

doi: 10.1371/journal.ppat.1013709

Figure Lengend Snippet: (A-B) C57BL/6J male mice were treated with diABZI (0.625, 1.25 and 2.5 mg/kg) or vehicle by either intraperitoneal (IP) injection or tail intravenous (IV) injection (A) . C57BL/6J mice were treated with GS-9620 (15 mg/kg) by gavage and DMXAA (12.5 and 25 mg/kg) or vehicle by intraperitoneal (IP) injection (B) . At 4 h after treatment, the serum levels of IFNβ were determined by ELISA (n = 4-5/group). *** P < 0.001,** P < 0.01, * P < 0.05 by One-way ANOVA. (C-O) C57BL/6J male mice were treated with diABZI (0.625, 1.25 and 2.5 mg/kg), DMXAA (20 mg/kg) or vehicle by IP injection, or GS-9620 (15 mg/kg) by gavage and sacrificed at 2 h after treatment. The mRNA levels of IFNβ, IFNα, TNFα, IL-6, CXCL10, IFIT1, IFITM3, ISG15, Mx1, Mx2, OAS1, OAS3 and STAT1 in the livers were determined by qRT-PCR (normalized to GAPDH) (n = 3-4/group). Fold induction of gene expression relative to that in vehicle-treated controls was presented as mean values ± standard deviations (SD). With the exception of IFITM3, the others were subjected to a natural logarithm transformation (ln transformation), and then a One-way ANOVA analysis was performed. *** P < 0.001,** P < 0.01, * P < 0.05.

Article Snippet: Diamidobenzimidazole (diABZI) (cat no. S8796) and DMXAA (cat no. S1537) were purchased from Selleck.

Techniques: Injection, IV Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Transformation Assay

Journal: PLOS Pathogens

Article Title: Antiviral and immune modulatory activities of STING agonists in a mouse model of persistent hepatitis B virus infection

doi: 10.1371/journal.ppat.1013709

Figure Lengend Snippet: (A) The STING protein level in the spleen of hSTING mice was detected by a Western blotting assay with an anti-STING antibody which can detect both human and mouse STING. (B-C) Effect of DMXAA and diABZI on the expression of CXCL10, IFIT1, ISG15, Mx1 and Mx2 in splenocytes and PBMCs from hSTING mice. The amounts of mRNAs specifying the specific cytokines and ISGs were quantified by qRT-PCR assay. Data are expressed as fold induction of gene expression relative to that in negative control (NC) treated with DMSO. *** P < 0.001,** P < 0.01, * P < 0.05 by Student’s t -test. (D-I) Experimental setup for in vivo efficacy study in humanized STING mice (D). All mice received a single i.v. injection of 8 × 10 10 vg of rAAV8-HBV1.3. On day 52 after inoculation, mice were grouped and treated with 2 mg/kg of diABZI or 20 mg/kg of DMXAA. Mice treated with vehicles served as control. Images of the mouse, syringe, and liver were sourced from https://openclipart.org/17558 , 282069, and 37315, respectively. (E) Body weight of mice before and after drug treatment is presented. (F-H) Serum levels of HBV DNA, HBsAg and HBeAg were determined by qPCR or ELISA on days 39 (before treatment), 54, and 61 after rAAV-HBV injection. *** P < 0.001 by Two-way ANOVA. (I) Liver HBV DNA levels were determined by qPCR (normalized per 1 μg of total liver DNA). *** P < 0.001 by One-way ANOVA. (J) Effect of DMXAA and ETV on serum levels of HBV DNA in the rAAV-HBV transduced WT mice. C57BL6/J mice injected with 8 × 10 10 vg of rAAV8-HBV1.3 genotype D by the tail vein for 8 weeks were divided into three groups (n = 6/group). Mice were then administered with a single dose of vehicle or DMXAA (25 mg/kg) via IP. Mice were also given ETV (0.1 mg/kg) by gavage daily. After one week posttreatment, the levels of serum HBV DNA in three groups of mice were measured by qPCR assay.*** P < 0.001,* P < 0.05 by One-way ANOVA.

Article Snippet: Diamidobenzimidazole (diABZI) (cat no. S8796) and DMXAA (cat no. S1537) were purchased from Selleck.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Gene Expression, Negative Control, In Vivo, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STNG promotes Z-NA accumulation and ZBP1-mediated PANoptosis. A Venn diagram showing shared regulatory genes in the indicated pathways. Gene list derived from RNA-seq data in Figure . B RNA-seq Transcripts per million (TPM) values showing the expression of selected RNAs. ZBP1 is the most significantly upregulated gene involved in PANoptosome formation, comparing CTRL and HSV-1-infected groups in WT retinas. C WB shows indicated protein levels in mouse retinas. Mice were IVT injected with 4x10^4 PFU HSV-1 and analyzed at 3 dpi. D IF of primary microglia (PMs) infected with HSV-1 (multiplicity of infection (MOI) = 10, 6 h) showing HSV-1 gB (red) and Z-NA (purple). Scale bars: 100 μm. n = 4 independent cultures. E Left: Percentage of HSV-1-positive cells. Right: Percentage of Z-NA-positive cells. Each dot represents an independent experiment.Left: Percentage of HSV-1-positive cells. Right: Percentage of Z-NA-positive cells. Each dot represents an independent experiment. F - I BV2 Cells were treated without or with STING agonist DMXAA (20 μg/mL) in combination with PBS, or DNase I (10 μg/mL), or RNase A (100 U/mL) for 6 h before analysis. F IF analysis showing Z-NA and ZBP1. Scale bars: 5 μm. G Quantification of Z-NA specks per cell. Each dot represents a value from randomly selected cells in two independent experiments. H Propidium iodide (PI) staining shows dead cells under the indicated treatment. Scale bar: 50 μm. I Percentage of dead cells. Each dot represents an independent experiment. J ScRNA-seq showing ISG genes with A-I high editing potential in retinal microglia. K IF analysis of STING, ZBP1 and Z-NA in BV2 cells. Cells were infected without or with HSV-1 ((MOI=10) and analyzed 2 h post-infection. Scale bars: 5 μm L Intensity profiles in each channel along the white line in the merged image. M Quantification of STING-Z-NA-ZBP1 speck area. Each dot represents a value from a randomly selected speck in two independent experiments. Results are presented as mean ± SD. Statistical analysis: panels (B, E, G, I, M), two-way ANOVA followed by Tukey’s post hoc test. (*p < 0.05; **p < 0.01; ****p < 0.0001)

Article Snippet: Cells were treated with 20 μg/mL STING agonist DMXAA (Selleck) in combination with PBS, 10 μg/mL DNase I, or 100 U/mL RNase A for 6 h. Vero cells were cultured in Vero-specific medium and used for plaque assays to determine viral titers.

Techniques: Derivative Assay, RNA Sequencing, Expressing, Infection, Injection, Staining