Journal: PLoS ONE

Article Title: Type-I Interferon is Critical for FasL Expression on Lung Cells to Determine the Severity of Influenza

doi: 10.1371/journal.pone.0055321

Figure Lengend Snippet: B6 mice were intranasally infected with 10 5 (closed triangle) or 10 2 (open square) pfu/head of the PR/8 virus. At 0, 3 or 5 DPI, the BALF of these mice were isolated. The amount of IFN- ß or total protein contained in these samples was assessed by mouse IFN- ß specific ELISA or BCA protein assay, respectively. The amounts of IFN- ß were normalized by that of the total protein in each sample. “N.D.” means not detected.

Article Snippet: The amount of IFN-β was assessed by mouse IFN-beta ELISA kit (R&D systems, Abingdon, UK).

Techniques: Infection, Virus, Isolation, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay

Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Heatmap of bulk RNA-seq in HT1080 cells treated with 100 µM BT versus DMSO for 12 hours. (B) Gene set enrichment analysis (left) and over-representation analysis (right) of BT-regulated genes, highlighting significant enrichment of antiviral and innate immunity pathways (GO and KEGG). (C) Volcano plot of differential expression in BT-versus DMSO-treated HT1080 cells (|log₂FC|>1, FDR<0.05). (D) RT-qPCR quantification of Ifnb1 and Oas2 mRNA in mouse bone marrow–derived macrophages (BMDMs) treated with indicated concentrations of BT for 12 hours. (E) Dose-dependent induction of IFNB1 mRNA (RT-qPR, top panels) and corresponding protein responses (western blots, bottom panels) in RAW 264.7, HT1080, HT29 and HeLa cells treated with the indicated BT concentrations for 12 hours. (F) RT-qPCR of IFNB1 and OAS2 in human PBMCs. (G) In vivo induction of Ifnb1 and Oas2 mRNA in heart, liver, lung, spleen and kidney of C57BL/6J mice 6 hours after a single intraperitoneal injection of BT (50 mg/kg). (H) RT-qPCR of Ifnb1 in RAW 264.7 cells following BT treatment (50, 75, 100 µM) for 12 hours in wild-tpye and knockout cells. Western blots showed absence of Viperin, IFIT3 and OAS2 induction in TBK1 knockout cells. (I) Flow cytometry of VSV-GFP infection in WT and TBK1 knockout RAW 264.7 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. Numbers indicated percentage of GFP positive cells. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Secreted IFN-β levels were measured using a commercial human IFNB1 ELISA Kit (BOSTER, EK2286) per the manufacturer’s protocol.

Techniques: RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Derivative Assay, Western Blot, In Vivo, Injection, Knock-Out, Flow Cytometry, Infection

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Predicted binding affinity scores of BT against pattern recognition receptors using the DeepAVC model. (B) SPR sensorgrams of BT binding to recombinant human cGAS at the indicated concentrations (0.196-50 µM). (C) Dose–response curve fitted from SPR data in (B). (D) RT-qPCR of IFNB1 mRNA in wild-type (WT), cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75 or 100 µM) or DMSO for 12 h. (E) Secreted type I IFN measured by ELISA in the same cell lines and treatments as in (D). (F) Flow cytometry of VSV-GFP infection in WT, cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. (G) Western blots of WT, cGAS knockout and STING knockout HT1080 cells treated as in (D). (H-I) In vivo VSV and HSV-1 challenges in cGAS knockout mice. Left panel is survival curves of mice, right panel is viruses RNA levels in liver measured by RT-qPCR. (J) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for DAPI (blue) and cytosolic dsDNA (green). Scale bars, 5 µm. (K) Confocal images of RAW 264.7 cells treated as in (J), stained with DAPI (blue) and MitoTracker (magenta) to visualize mitochondrial morphology. Scale bars, 5 µm. (L) Comparison of mitochondrial structure by confocal versus STED super-resolution microscopy in RAW 264.7 cells treated with DMSO or BT and stained with PK Mito. Scale bars, 2 µm. (M) Transmission electron micrographs of mitochondria in RAW 264.7 cells treated with DMSO or BT. (N) In vitro cGAS enzymatic assays: LC-MS quantification of cGAMP production by recombinant cGAS incubated with dsDNA in the presence of BT (25 or 100 µM). (O) LC-MS analysis of cGAMP production by cGAS incubated with BT (25 or 100 µM) in the absence of exogenous DNA. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Secreted IFN-β levels were measured using a commercial human IFNB1 ELISA Kit (BOSTER, EK2286) per the manufacturer’s protocol.

Techniques: Binding Assay, Recombinant, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Infection, Western Blot, In Vivo, Staining, Comparison, Super-Resolution Microscopy, Transmission Assay, In Vitro, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) RT-qPCR of HDAC mRNA in HT1080 cells treated with 100 µM BT or DMSO for 12 hours. (B) Fluorometric HDAC activity assay in HT1080 cells after 12 hours treatment with BT (50, 75, 100 µM) or DMSO. Enzymatic activity is shown as percentage of DMSO control. (C) Western blot of global H3K27ac levels in HT1080 cells. (D) CUT&Tag metaprofile (top) and heatmap (bottom) of H3K27ac signal ±1 kb around called peaks. (E) ATAC-seq metaprofile (top) and heatmap (bottom) of chromatin accessibility ±1 kb around peak centers. (F) Genome browser (IGV) snapshots at MX1 and IFIT1 loci showing H3K27ac CUT&Tag and ATAC-seq. (G) Venn diagram of overlapping regions showing increased H3K27ac, increased accessibility and increased gene expressions in BT-treated cells. (H) CUT&Tag metaprofile (top) and heatmap (bottom) of TRIM28 occupancy ±1 kb around called peaks, illustrating widespread loss of TRIM28 binding. (I) Boxplots in CUT&Tag signal for H3K27ac (left) and TRIM28 (right) at consensus peak regions. Time-course CUT&Tag-qPCR at an OAS2 and IFNB1 regulatory locus, measured at 0, 4, 8 and 12 h after BT (100 µM) treatment. (K) Heatmap of the top 40 up-regulated ERV families in bulk RNA-seq. (L-N) Meta-profiles of chromatin changes at the set of BT-reactivated ERV loci (±2 kb from center). (L) H3K27ac CUT&Tag; (M) TRIM28 CUT&Tag; (N) ATAC-seq heatmaps. (O) Representative IGV tracks at the HERVK14-int locus showing BT-induced gain of H3K27ac (top), loss of TRIM28 binding (middle) and emergence of an ATAC-seq peak (bottom). (P) Four-way Venn diagram of ERV loci identified by bulk RNA-seq up-regulation, H3K27ac CUT&Tag gain, TRIM28 CUT&Tag loss and increased ATAC-seq accessibility. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Secreted IFN-β levels were measured using a commercial human IFNB1 ELISA Kit (BOSTER, EK2286) per the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, HDAC Activity Assay, Activity Assay, Control, Western Blot, Binding Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Distribution of transposable element (TE) classes among loci up-regulated by BT in bulk RNA-seq (counts of LTR, DNA, LINE, SINE, Satellite and Unknown elements). (B) Reactome terms enriched among BT-reactivated ERV loci. (C) GSEA enrichment plots in BT-versus DMSO-treated HT1080 cells. (D) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for dsRNA (green) and nuclei (DAPI, blue). BT treatment induces punctate cytosolic dsRNA accumulations. Scale bars, 5 µm. (E) RT-qPCR of IFNB1 mRNA in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (F) Secreted type I IFN measured by ELISA in the same HeLa cell lines and treatments as in (E). (G) Western blot of p-TBK1, total TBK1, MDA5, MAVS and RIG-I, the samples were same to (E). (H) Flow cytometric quantification of VSV-GFP infection in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (I-J) Left are survival curves of MAVS knockout C57BL/6J mice challenged with lethal VSV and HSV-1 after a single i.p. dose of BT (50 mg/kg). Right are viruses RNA levels in livers measured by RT-qPCR. RIP-qPCR detection of HERVK14-int RNA associated with RIG-I or MDA5 in RAW 264.7 cells treated with DMSO or BT for 12 h. (L) ELISA quantification of secreted type I IFN and RT-qPCR of IFNB1 mRNA. (M) Flow cytometry of VSV-GFP infection. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Secreted IFN-β levels were measured using a commercial human IFNB1 ELISA Kit (BOSTER, EK2286) per the manufacturer’s protocol.

Techniques: RNA Sequencing, Staining, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Flow Cytometry

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: ( A ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points, and then cell lysates were analyzed for EMCV replication level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000039 (4 h), *** P = 0.00050 (8 h), **** P = 0.000040 (12 h). ( B ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points. The cell supernatant was harvested and analyzed by TCID 50 assay. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00540 (8 h), ** P = 0.00820 (12 h). ( C ) Western blot analysis of the indicated signaling proteins in MEFs from Rnf144b +/+ or Rnf144b −/− mice infected with EMCV (MOI = 1) for the indicated time periods. ( D ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). After 12 h, cell lysates were analyzed for Ifnb1, TNF-α, IL-6 mRNA level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000009 (Ifnb1), *** P = 0.00090 (TNF-α), *** P = 0.00030 (IL-6). ( E ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). 12 h post-infection, cells were treated by CHX (30 μM) for indicated time points. Protein extracts were used for immunoblot analysis of the endogenous MDA5 protein level. ( F , G ) Rnf144b +/+ and Rnf144b −/ − mice were intraperitoneally injected EMCV (5 × 10 6 PFU) for 48 h ( n = 5 per group). qPCR analysis of EMCV replication level in heart and brain, and mRNA level of Ifnb1, TNF-α, IL-6 in brain ( G ). Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00530 (heart), * P = 0.02620 (brain), *** P = 0.00030 (Ifnb1), ** P = 0.00170 (TNF-α), *** P = 0.00030 (IL-6). ( H ) ELISA of IFN-β production in serum of Rnf144b +/+ and Rnf144b −/− mice that were intraperitoneally injected with EMCV (5 × 10 6 PFU) for 12 h ( n = 3 per group). Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000020. ( I ) Survival of Rnf144b +/+ and Rnf144b −/− mice ( n = 20 per group) intraperitoneally infected with EMCV (6 × 10 7 PFU). Significance was tested using log-rank (Mantel-Cox) test, *** P = 0.00030. Data information: Data shown are representative of at least three biological replicates, with each data point representing a biological experiment. Error bars are presented as mean ± SD. Statistical significance was determined by Student’s t-test. .

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Infection, Two Tailed Test, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Recombinant, Sequencing, shRNA, Mutagenesis, RNA Extraction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: HSV1 recombinant viruses expressing cGAS and/or STING exhibit restricted replication in human cancer cells (A) Schematic diagram of rHSV1 constructs. (B–N) 2 × 10 5 293T, hTERT, HT29, and SW48 cells infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at the MOI indicated. (B) Immunoblot analysis of cGAS, STING, phospho-STING, phospho-TBK1, phospho-IRF3, and β-actin 6 h post infection, (C) percentage of viable cells ( n = 4 biological replicates), and (D) virus titers ( n = 2 biological replicates). (E) Measurement by ELISA of the quantity of 2′3′ cGAMP in 5 × 10 5 293T cells 24 h post infection ( n = 3 biological replicates). (F) IFN-β-luciferase activity in 293T cells 24 h after plasmid transfection followed by 6 h of infection ( n = 3 technical replicates). (G, I, and K) Percentage of viable cells ( n = 3 biological replicates) and (H, J, and L) virus titers ( n = 3 biological replicates) on infected hTERT, HT29, and SW48 cells at MOI 1. (M) qPCR of Cxcl10 ( n = 2 biological replicates) and (N) ELISA analysis of human IFNβ production in hTERT, HT29, and SW48 cells 24 h after infection ( n = 6 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Recombinant, Expressing, Construct, Infection, Western Blot, Virus, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Recombinant HSV1 exhibits diminutive oncolytic activity yet retains in vivo anti-tumor properties dependent on extrinsic STING signaling (A) Immunoblot analysis of cGAS, STING, and β-actin in B16-OVA, B16-OVA cGAS KO (CKO), B16-OVA STING KO (SKO), and B16-OVA STING/cGAS KO (S/CKO) cells. (B and C) 2 × 10 5 B16 cells were infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at MOI 5 for 24 h. (B) Virus titers ( n = 2 biological replicates) and (C) percentage of viable cells were measured ( n = 2 biological replicates). (D–F) 2 × 10 5 293T and B16-OVA cells were infected at MOI 0.1 or 5 with HSV1- Δγ34.5-GFP. (D) The virus titer was determined by plaque assay ( n = 3 biological replicates), (E) the percentage of GFP + cells were measured by cytometry ( n = 3 biological replicates), and (F) the quantification of HSV1- Δγ34.5 genome was done by qPCR at 3 or 6 h, 24 h, and 48 h post infection ( n = 3 biological replicates). (G) B16 cells were infected with HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A at MOI 5 for 6 h. B16-OVA cells were treated with 3 μg/mL dsDNA90 as a control, and Cxcl10 was analyzed by qPCR ( n = 4 technical replicates). (H–Q) Wild-type (H–J: n = 6–8 mice per group; L–N: n = 4 mice per group), STING KO C57BL/6J ( n = 11–12 mice per group on 2 independent experiments), and BALB/c nude mice ( n = 7 mice by groups) were subcutaneously injected as indicated with B16-OVA, B16-OVA CKO, or B16-OVA S/CKO cells on the flank (5 × 10 5 cells/mouse). 5 × 10 6 PFU of replicating HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A was injected intratumorally (black arrows) three times. (H–L and O) The tumor volume was measured on the indicated days and calculated with the formula V = (length × width 2 )/2. At 16 or 17 days, the spleen and the tumors were extracted. (M and P) Digital photograph of tumors and (N and Q) ELISpot to measure IFNg release from CD8 + T cells. (R and S) Phagocytosis of B16-OVA and B16-OVA S/CKO cells by murine WT and STING KO macrophages. 1 × 10 6 cells were infected with HSV1-Δγ34.5 for 40 h at MOI 20 then irradiated by UV (120 mJ/cm) and incubated for 24 h. The irradiated cells were fed to macrophages (MØ) (2 × 10 5 cells). (R) Schematic representation and (S) ELISA analysis of IFN-β at 24 h in macrophages following engulfment of B16 ( n = 5 [WT macrophages] and 3 [STING macrophages] technical replicates). Error bars indicate mean ± SEM; Student’s t test and (H–L and O) ordinary one-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Recombinant, Activity Assay, In Vivo, Western Blot, Infection, Virus, Plaque Assay, Cytometry, Injection, Enzyme-linked Immunospot, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Murine and human tumor cells exposed to nano-STAVs activate APCs in trans in an STING-dependent manner, augmenting checkpoint therapeutic activity in vivo (A) Schematic representation of the phagocytosis of mouse and human cells by macrophages. 1 × 10 6 cells were treated with 1 μg/mL of nano-empty or nano-STAVs or transfected with lipofectamine + STAVs and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine or human macrophages (MØ) (2 × 10 5 cells) 24 h after UV irradiation. (B) Confocal microscopy analysis with nano-STAVs-cy5 (red) in CD11b + FITC murine macrophages (green). Cells were treated for 6 h with nanoparticles, and the phagocytosis was evaluated at 6 h. (C–H) (C, E, and G) RT-qPCR analysis of Cxcl10 at 6 h and (D, F, and H) IFN-β ELISA at 24 h in human and murine WT macrophages following engulfment of B16, SK-MEL-31, and SK-MEL-5 cells in presence or absence of nano-STAVs for 24 h ( n = 4 [mouse cell] and 3 [human cells] biological replicates). (I–L) Mice were subcutaneously injected with B16-OVA cells (5 × 10 5 cells/mouse) ( n = 13–17 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) (black arrows). At day 19, the spleen was extracted to measure IFNg release from CD8 + T cells. (I) Schematic representation of experimental design. (J) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Digital photographs of tumors. (L) IFNg ELISpot. (C–H) Error bars indicate mean ± SEM; Student’s t test and (J and L) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Activity Assay, In Vivo, Transfection, Irradiation, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs activity is augmented by type I IFN (A–D) Mice were subcutaneously injected with B16 OVA cells (5 × 10 5 cells/mouse) ( n = 17–18 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) and/or IFNa (10,000 U/mouse) (black arrows). At day 17, the spleen was extracted to measure IFNg release from CD8 + T cells. (A) Schematic representation of experimental design. (B) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (C) IFNg ELISpot. (D) Digital photographs of tumors. (E–J) qPCR analysis of CXCL10 and CCL5 in B16-OVA, B16-OVA-cGAS KO (CKO), and WT and SKO murine macrophages (2 × 10 5 cells), treated with IFNa at 1000 U/ml for 6 h ( n = 4 biological replicates). (K and L) Flow cytometry for H-2Kb and CD86 on macrophages (2 × 10 5 cells) following IFNa treatment at 1000 U/ml for 24 h ( n = 2 biological replicates). (M) Schematic representation of the phagocytosis of B16-OVA cells by macrophages. 1 × 10 6 B16-OVA cells were treated with IFNa at 1000 U/ml for 24 h and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine macrophages (MØ) (2 × 10 5 cells) previously treated or not with IFNa at 1000 U/ml for 24 h. (N) RT-qPCR analysis of Cxcl10 at 6 h ( n = 2 biological replicates). (O) IFN-β ELISA at 24 h of murine WT macrophages following engulfment of B16-OVA treated with IFNa ( n = 3 biological replicates). (P) Schematic representation of the phagocytosis of untreated B16-OVA cGAS KO cells (B16 CKO) by macrophages previously treated with IFNa at 1000 U/ml for 24 h. The conditions applied were the same as in (M). (Q) Flow cytometry for H-2Kb-SIINFEKL (OVA) on macrophages at 24 h following phagocytosis of B16-OVA cGAS KO ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test and (B and C) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Activity Assay, Injection, Enzyme-linked Immunospot, Flow Cytometry, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet:

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Staining, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Knock-Out, Plasmid Preparation, Expressing, Software, Microscopy