Journal: JCI Insight

Article Title: Coagulation proteases modulate nucleic acid uptake and cGAS-STING-IFN induction in the tumor microenvironment

doi: 10.1172/jci.insight.190311

Figure Lengend Snippet: ( A ) EdU labeled cell free tumor debris uptake for 2 hours in BM monocyte derived macrophages from PAR2 WT /F10 fl/fl , PAR2 G37I , or F10 fl/fl LysMcre mice determined by flow cytometry; n = 3–6, 1-way ANOVA with Dunnett’s multiple comparison test. ( B ) Intracellular cGAMP produced by macrophages from F10 fl/fl or F10 fl/fl LysMcre mice after tumor debris loading for 6 or 16 hours versus untreated controls; n = 4, 2-way ANOVA with Sidak’s multiple comparison test. ( C ) Western blot showing phospho-IRF3 (IRF3-P), IRF3, and FX protein expression by macrophages from F10 fl/fl or F10 fl/fl LysMcre mice with loading control β actin. Cells were either untreated or loaded with tumor debris or polyI:C (25 μg/mL) as positive controls for phosphor-IRF3 for 90 minutes. Lanes were run on the same gel but were noncontiguous. ( D ) Mb21d1, Sting1, and Ifnb1 mRNA expression normalized to r18s by PAR2 WT /F10 fl/fl , PAR2 G37I or F10 fl/fl LysMcre derived macrophages with and without tumor cell debris loading for 2 hours; n = 3–12, 2-way ANOVA with Sidak’s multiple comparison test. ( E ) Tumor debris uptake determined by flow cytometry of iDCs from PAR2 WT , PAR2 G37I , F10 fl/fl , or F10 fl/fl LysMcre; n = 5–7, 1-way ANOVA with Dunnett’s multiple comparison test. ( F ) Mb21d1, Sting1, Ifnb1, Itgae and Il12p40 mRNA expression normalized to r18s by PAR2 G37I , F10 fl/fl vs F10 fl/fl LysMcre derived iDCs with and without tumor debris loading for 2 hours; n = 3–6, 2-way ANOVA with Sidak’s multiple comparison test. * P < 0.05 ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Membranes were blocked with milk powder TBST and incubated over night with anti-mouse prothrombin (abcam AB208590 ; 1:1000), anti-mouse FX (Ruf lab; 1:500), anti-mouse FV(a) (GMA-753 Green Mountain; 1:1000), anti-mouse IRF3 (D83B9, #4302, Cell Signaling; 1:1000), anti-mouse phospho-IRF3 (Ser396) (4D4G, #4947, Cell Signaling; 1:1000), and anti-mouse β actin (8H10D10, #3700, Cell Signaling; 1:1000) in 5% dry milk-TBST, washed and incubated with the secondary antibody anti-rabbit HRP conjugated (# 7074, Cell Signaling, 1:5000) or anti-rat HRP (# 7077 Cell signaling, 1:5000), and finally developed with ECL reagent (SIRIUS).

Techniques: Labeling, Derivative Assay, Flow Cytometry, Comparison, Produced, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. Chemical structures of INI3067, INI3069, INI3070, and INI3071. THF-ISRE cells deficient in TRIF/MAVS or STING were exposed in duplicate overnight to G10 or analogs INI3067, INI3069, INI3070, or INI3071 at indicated concentration; B. IRF3/IFN-I-dependent luciferase expression determined by measuring luminescence. Data presented are mean ± SEM relative luminescence units (RLU) determined in relation to dosage matched DMSO-treated cells; C. Cell viability was determined using ATP-based Cell Titer Glo assay. Data presented are raw luminescence values; D. THF-MAVS/TRIF -/- or THF-STING -/- cells were exposed in duplicate for 6 h to DMSO, 1000 HAU SeV, 100 µM AV-C, 100 nM diABZI, or 100 µM G10, INI3067, INI3069, INI3070, or INI3071. Transcription of mRNAs encoding CXCL10, Viperin, or IFIT1 were then determined using qPCR. Data presented are mean ± SD fold changes of indicated transcript relative to DMSO treated cells; E. THF-MAVS/TRIF -/- or THF-STING -/- cells were exposed for 4 h to DMSO, 1000 HAU SeV, 100 µM AV-C, 100 nM diABZI, or 100 µM G10, INI3067, INI3069, INI3070, or INI3071 and immunoblot of whole cell lysates used to determine expression and phosphorylation status of STING, IRF3, and TBK1 with GAPDH as a loading control; F. HEK293T cells were left untransfected or transiently transfected for 24 h with vectors expressing the WT, HAQ, or R232H alleles of human STING. Cells were then exposed to DMSO, 100 nM diABZI, or 100 µM INI3067, INI3069, INI3070, or INI3071 for 4 h and immunoblot used to determine expression levels of STING and IRF3 and determine the phosphorylation status of IRF3 with GAPDH as a loading control; G. HEK293T cells were stably transfected with indicated STING alleles and transiently transfected with an IRF3/IFN-I-responsive LUC reporter vector and exposed in duplicate overnight to indicated concentrations of INI3067, INI3069, INI3070, or INI3071 or dosage matched DMSO. Data displayed are mean ± SEM RLU.

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: Concentration Assay, Luciferase, Expressing, Glo Assay, Western Blot, Phospho-proteomics, Control, Transfection, Stable Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. THF cells were treated in duplicate for 6 h with INI3067, INI3069, INI3070, or INI3071 or dosage-matched DMSO and infected with CHIKV-nLuc at MOI = 0.1. At 24 h post infection nLuc expression was measured as luminescence. Data presented are mean ± SEM infection levels indicated as percentage of luminescence signal relative to that generated by DMSO control; B . THF cells lacking MAVS/TRIF, STING, IRF3, or ATG5 were treated in duplicate for 6 h with INI3069 at indicated concentration (left), DMSO, or 100U IFNb (right) and infected with CHIKV-nLuc and luminescence measured and displayed as in A; C. Immunoblot showing expression of ATG5 in THF or ATG-deficient THF.

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: Infection, Expressing, Generated, Control, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. 6xHIS-tagged cytosolic domain of STING was synthesized in BL21 bacteria and purified. It was then untreated or mixed with 50 µM INI3069 or 100 µM cGAMP in the presence of SYPRO orange. Fluorescence was measured over the indicated temperature gradient. Data displayed are fluorescence signal for indicated treatments at indicated temperatures; B. HEK293T cells were transiently transfected overnight with vectors encoding the WT human STING allele or STING coding regions containing alanine mutations in the TMD (L30A, Y104A) as indicated. Cells were then exposed for 4 h to 0.5% DMSO, 10 nM diABZI, 100 µM C53, or 6.25 µM INI3069. SDS-PAGE was then used on whole cell lysates and stained for phosphorylated IRF3 or STING; C. THF-MAVS/TRIF-/- cells were treated 3 h with DMSO, cGAMP, C53, or INI3069. SDS-PAGE was then used on whole cell lysates and stained for phosphorylated TFEB, total TFEB, phosphorylated IRF3, total IRF3, or b-Actin.

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: Synthesized, Bacteria, Purification, Fluorescence, Transfection, SDS Page, Staining

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. THF-MAVS/TRIF -/- cells were treated 3 h with DMSO, cGAMP, C53, or INI3069. SDS-PAGE was then used on whole cell lysates and stained for phosphorylated TFEB, total TFEB, phosphorylated IRF3, total IRF3, or b-Actin; B and C. HEK293T cells were stably transduced with GFP-mCherry-LC3 and two single cell clones transfected for 24 h with a plasmid encoding WT human STING or left untransfected. They were then exposed for 4 h to DMSO, 1 µM diABZI, 10 µM C53, or 10 µM INI3069 and flow cytometry used to quantify intracellular expression of mCherry and GFP. B. Summary of mCherry:GFP MFI ratio for individual cells (excluding the top and bottom 10% of observations); C. Representative designations of degree of autophagic flux as determined by mCherry:GFP ratio between treatments. Strategy for designating low, intermediate and high autophagic flux based on mCherry:GFP MFI ratio (top). Data presented are proportion of cells displaying indicated level of autophagic flux following indicated treatment in the presence and absence of transfected STING (bottom). Experiments were performed in duplicate for both clonal cell lines and one way ANOVA with Dunnet’s correction for multiple comparisons used to determine statistical significance of cell populations designated as displaying high autophagic flux (***p < 0.001).

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: SDS Page, Staining, Stable Transfection, Transduction, Clone Assay, Transfection, Plasmid Preparation, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. MEFs from indicated strain were treated ex vivo with DMSO, 100 HAU SeV, 100 nM diABZI, 50 µM DMXAA, or 50 µM INI3069 for 4 h and immunoblot used to measure expression and phosphorylation status of STING, TBK1, or IRF3 with bActin as loading control; B. Splenocytes were harvested and treated ex vivo from indicated mouse strain and exposed in duplicate overnight to DMSO, or 50 µM DMXAA, G10, or INI3069. Secretion of CXCL10 was then measured using Luminex assay. Data presented are mean ± SD levels detected in media; C. MEFs from indicated strain were exposed in duplicate to DMSO, 100 µM G10, 100 µM INI3069, 100 nM diABZI, or 50 µM DMXAA for 6 h and qPCR used to measure transcript levels of CXCL10, IFIT1, or Viperin. Data presented are mean ± SD mRNA fold changes relative to DMSO treated cells. Student’s t-test was used to compare expression relative to DMSO treatment (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: Ex Vivo, Western Blot, Expressing, Phospho-proteomics, Control, Luminex

Journal: bioRxiv

Article Title: Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

doi: 10.1101/2025.07.14.664814

Figure Lengend Snippet: A. Immature BMDC were generated from bone marrow of indicated strain as described in text and treated in duplicate for 24 h with DMSO, 10 nM diABZI, or 10 µM INI3069. Flow cytometry was then used to measure surface expression of CD40, CD80, and CD86. Data presented are sample flow cytometry plots for individual samples (top) and mean ± SD MFI for indicated markers (bottom). One way ANOVA was used to determine significance (**p < 0.01, ***p < 0.001, ****p < 0.0001); B. BMDC from huSTING mice were treated 3 h with DMSO, cGAMP, C53, or INI3069. SDS-PAGE was then used on whole cell lysates and stained for phosphorylated TFEB, total TFEB, phosphorylated IRF3, total IRF3, or b-Actin.

Article Snippet: Anti-mouse β-Actin (# 5125), anti-mouse/human STING (# 13647), anti-mouse phospho-S366 STING (# 72971), anti-human phospho-S365 STING (# 19781), anti-mouse/human IRF3 (# 4302), anti-mouse phospho-S379 IRF3 (# 79945), anti-mouse/human TBK1 (# 3504), anti-mouse/human phospho-S172 TBK1 (# 5483), and anti-phospho-S211 TFEB (# 37681S) were purchased from Cell Signaling.

Techniques: Generated, Flow Cytometry, Expressing, SDS Page, Staining