Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative Airyscan-processed confocal images of HeLa STING and HeLa STING cells with stable overexpression of mEGFP-LC3B (green) treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta) and STING (cyan). Scale bar = 20 and 2 µm (inset). Imaging was replicated in two independent experiments. ( B ) Representative immunoblots of indicated proteins detected lysates from HeLa STING WT and HeLa STING with stable overexpression of mEGFP-LC3B prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in 3 independent experiments. ( C ) Representative immunoblots of indicated proteins detected in HeLa STING cell lysates prepared after treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( D , E ) Representative spinning disk confocal images of FRT/TREX HeLa cells stably expressing FRT/TO-DD-Vx3-EGFP, BFP-P2A-STING, and mScarletI-LC3B at the 6-hour timepoint following treatment ( D ) and quantification of the percentage (%) of cells positive for Vx3-EGFP foci over time ( E ). Cells were incubated with 1 µg/mL Doxycycline and 500 nM Shield1 for 24 h prior to treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI, and imaging every 30 min for 12 h on a spinning disk confocal microscope. Scale bar = 50 µm. Quantification is from three wells analyzed in the same experiment. Imaging was replicated in two independent experiments.

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Over Expression, Immunostaining, Ubiquitin Proteomics, Imaging, Western Blot, Stable Transfection, Expressing, Incubation, Microscopy

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and ATG16L1KO cells prepared following treatment with 1 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. ( B ) Representative immunoblots of indicated proteins detected in lysates from WT THP1 cells prepared following treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( C , D ) Relative expression changes of indicated NFκB-related genes ( C ) and interferon-related genes ( D ) detected by quantitative RT-PCR in THP1 cells treated with DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A one-way ANOVA with Tukey’s multiple comparisons test was performed on 2 −ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 ( TNF p = 0.0123; TNFAIP3 p = 0.005; IL6 p = 0.0027; IFNB1 p = <0.0001; ISG15 p = 0.0351).

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Recombinant, Ubiquitin Proteomics, Sequencing, CRISPR, shRNA, Reverse Transcription, SYBR Green Assay, Control, Magnetic Beads, Negative Control, Cloning, Modification, Software

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 elicits a type I IFN response in porcine cells. (A) Schematic representation of the STING-mediated type I IFN response. (B) WT, Sting –/– , Tbk1 –/– , Irf3 –/– , and Ifnar1 –/– PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with G10 at the indicated concentrations for 24 h. Total mRNA was then reverse-transcribed to cDNA and IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (C) WT, Sting –/– , and Ifnar1 –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in B . IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (D) WT and Sting –/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in B . The medium was then harvested and IFN-β secretion was quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (E) WT, Sting –/– , Tbk1 –/– , Irf3 –/– , and Ifnar1 –/– PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in B . ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (F) WT, Sting –/– , and Ifnar1 –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in B . ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (G) WT and Sting –/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were infected with PRV-QXX (MOI = 1) and simultaneously treated with G10 as in B . Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID 50 assays. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by one-way ANOVA.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Infection, Virus

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 stimulates Sp1-dependent p65 transcription in porcine cells. (A) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10 (0.6–20 μM), and LPS (1 μg/ml) for 24 h. Total mRNA was then reverse-transcribed to cDNA and P65 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (B) Diagrams of the P65 promoter and the various mutants, with the Sp1 binding sites indicated. (C) WT and Sting –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were transfected with 0.1 μg/well p65 –Luc variants and 0.02 μg/well pCMV-Renilla. At 24 h post transfection, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 6 h. p65 promoter activity was assessed with dual luciferase reporter assays. *** P < 0.001 determined by two-tailed Student’s t -test. (D) p65 promoter activity was assessed with dual luciferase reporter assays in WT and Sting –/– PK15 cells the same as in C. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (E) 3D4/21 and PK15 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were transfected with indicated siRNA for 48 h. Sp1 protein was assessed with immunoblotting analysis. β-actin served as loading control. (F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were transfected with indicated siRNA for 48 h. Then cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. Total mRNA was then reverse-transcribed to cDNA and P65 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *** P < 0.001 determined by two-tailed Student’s t -test. (G) WT and Sting –/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, 3D4/21 cells were transfected with siSp1-3 for 48 h. Then WT, Sting –/– , and siSp1-3 transfected 3D4/21 cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml). Sp1 ChIP assays were performed at 24 h post treatment. *** P < 0.001 determined by two-tailed Student’s t -test.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Two Tailed Test, Binding Assay, Transfection, Activity Assay, Luciferase, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 activates the NF-κB signaling pathway in porcine cells. (A) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. Translocation of P65 into the nucleus (DAPI) was assessed by immunofluorescence analysis with antibody against P65. Quantification of cells with nuclear localized P65 is shown on the right ( n = 30 cells). Scale bar, 10 μm. *** P < 0.001 determined by two-tailed Student’s t -test. (B) WT and Sting –/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated as in A . Phospho-P65 and P65 were assessed with immunoblotting analysis in the cytosol and nuclei fraction. β-actin (indicating cytosol) and Lamin B1 (indicating nuclei) served as loading controls. (C,D) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with G10 at the indicated concentration for 24 h. Total mRNA was then reverse-transcribed to cDNA and IL-1β (C) and IL-18 (D) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (E,F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were transfected with indicated siRNAs for 48 h. Then, cells were treated as in A . Total mRNA was then reverse-transcribed to cDNA and IL-1β (E) and IL-18 (F) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (G) P65 protein was assessed with immunoblotting analysis in WT, p65 –/– 1#, and p65 –/– 2# 3D4/21 and PK15 cells. β-actin served as loading control. (H,I) WT, p65 –/– 1#, and p65 –/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in A . Total mRNA was then reverse-transcribed to cDNA and IL-1β (H) and IL-18 (I) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Translocation Assay, Immunofluorescence, Two Tailed Test, Western Blot, Concentration Assay, Reverse Transcription, Quantitative RT-PCR, Expressing, Transfection, Control

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 promotes IL-1β and IL-18 secretion in porcine cells. (A,B) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at indicated concentrations for 24 h. The medium was then harvested and IL-1β (A) and IL-18 (B) secretion was quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (C,D) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were transfected with indicated siRNA for 48 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at indicated concentrations for 24 h. The medium was then harvested and IL-1β (C) and IL-18 (D) secretion was quantified by ELISA. *** P < 0.001 determined by two-tailed Student’s t -test. (E,F) WT, p65 –/– 1#, and p65 –/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in C . The medium was then harvested and IL-1β (E) and IL-18 (F) secretion was quantified by ELISA. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (G) WT and Sting –/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated as in C . The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β by immunoblotting analysis. (H) WT and Sting –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, Sting –/– 3D4/21 cells were transfected with plasmid for expression of STING-Flag plasmid (4 μg) for 24 h. Then, cells were treated as in A . The medium was then harvested and IL-1β and IL-18 secretion was quantified by ELISA. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 induces ASC oligomerization and Caspase-1 activation in porcine cells. (A) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10 5 per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations for 24 h. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown on the right ( n = 30 cells). Scale bar, 10 μm. *** P < 0.001 determined by two-tailed Student’s t -test. (B) WT and Sting –/– PK15 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated as in A. ASC oligomerization was assessed by immunoblotting analysis. (C,D) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations in the absence (PBS) or presence of Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (E,F) WT and Asc –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10 and LPS + Nig at the indicated concentrations for 24 h. The medium was then harvested and IL-1β (E) and IL-18 (F) secretion were quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (G,H) WT and Caspase-1 –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in E . The medium was then harvested and IL-1β (G) and IL-18 (H) secretion were quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Microscopy, Two Tailed Test, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10 activates the NLRP3 inflammasome in porcine cells. (A) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations for 24 h. Total mRNA was then reverse-transcribed to cDNA and NLRP3 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (B) WT and Sting –/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10 5 per well. On the next day, cells were transfected with plasmid for expression of NLRP3-Flag (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS + Nig (1 μg/ml + 2.5 μM) for 24 h. NLRP3 activation was assessed by immunofluorescence analysis with antibody against Flag. Scale bar, 10 μm. (C,D) WT, Sting –/– , Nlrp3 –/– , Asc –/– , and Caspase-1 –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10, LPS + Nig, LPS + ATP, and VX765 at the indicated concentrations for 24 h. The medium was then harvested and IL-1β (C) and IL-18 (D) secretion were quantified by ELISA. *** P < 0.001 determined by two-tailed Student’s t -test. (E) WT and Nlrp3 –/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10 5 per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated as in B . ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown on the right ( n = 30 cells). Scale bar, 10 μm. *** P < 0.001 determined by two-tailed Student’s t -test. (F) WT and Nlrp3 –/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated as in B . ASC oligomerization was assessed by immunoblotting analysis. (G) WT and Nlrp3 –/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations in the absence (PBS) or presence of Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (H) WT and Nlrp3 –/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated as in B . The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β, pro-Caspase-1, and cleaved Caspase-1 (P20) by immunoblotting analysis.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Activation Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: G10-mediated activation of the NLRP3 inflammasome requires potassium flux in porcine cells. (A–D) 3D4/21 (A,B) and PK15 (C,D) cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with G10 (20 μm), LPS + Nig (1 μg/ml + 2.5 μM), and CL097 (70 μM) in the absence or presence of KCl (10–80 mM) for 24 h. The medium was then harvested and IL-1β (A,C) and IL-18 (B,D) secretion were quantified by ELISA. * P < 0.05, ** P < 0.01 determined by two-tailed Student’s t -test. ns, no significance. (E) 3D4/21 cells were seeded in 12-well plates with coverslips at a density of 1 × 10 5 per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10 (20 μm), and LPS + Nig (1 μg/ml + 2.5 μM) in the absence (PBS) or presence of KCl (80 mM) and NaCl (10 mM) for 24 h. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown at the bottom ( n = 30 cells). Scale bar, 10 μm. *** P < 0.001 determined by two-tailed Student’s t -test. ns, no significance. (F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with G10 (20 μm) and LPS + Nig (1 μg/ml + 2.5 μM) as indicated in the absence (PBS) or presence of KCl (45 mM) and Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. *** P < 0.001 determined by two-tailed Student’s t -test. (G) 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10 5 per dish. On the next day, cells were treated with vehicle (DMSO), G10 (20 μm), and LPS + Nig (1 μg/ml + 2.5 μM) in the absence (PBS) or presence of KCl (80 mM) for 24 h. The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β, pro-Caspase-1 and cleaved Caspase-1 (P20) by immunoblotting analysis.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transfection, Plasmid Preparation, Expressing, Fluorescence, Microscopy, Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: Inhibition of the NLRP3 inflammasome augments G10-induced type I IFN and antiviral activity in porcine cells. (A) WT, p65 –/– 1#, and p65 –/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. ** P < 0.01, *** P < 0.001 determined by one-way ANOVA. (B,C) 3D4/21 (B) and PK15 (C) cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with G10 (20 μM), MCC950 (10 μM), and VX765 (10 μM) as indicated for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by one-way ANOVA. (D,E) WT, Sting –/– , Nlrp3 –/– , Asc –/– , Caspase-1 –/– , p65 –/– 1#, and Ifnar1 –/– 3D4/21 (D) and WT, Sting –/– , Nlrp3 –/– , Asc –/– , Caspase-1 –/– , p65 –/– 1#, and Ifnar1 –/– PK15 (E) cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated with vehicle (DMSO) or G10 (20 μM) for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by one-way ANOVA. ns, no significance. (F,G) 3D4/21 (F) and PK15 (G) cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were infected with PRV-QXX (MOI = 1) and simultaneously treated as in B . Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID 50 assays. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by one-way ANOVA. (H,I) WT, Sting –/– , Nlrp3 –/– , Asc –/– , Caspase-1 –/– , p65 –/– 1#, and Ifnar1 –/– 3D4/21 (H) and WT, Sting –/– , Nlrp3 –/– , Asc –/– , Caspase-1 –/– , p65 –/– 1#, and Ifnar1 –/– PK15 (I) cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in D . Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID 50 assays. * P < 0.05, ** P < 0.01 determined by one-way ANOVA.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Virus

Journal: Frontiers in Immunology

Article Title: The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

doi: 10.3389/fimmu.2020.575818

Figure Lengend Snippet: Schematic model showing G10 activation of the NLRP3 inflammasome in porcine cells. In human cells, G10 activates only type I IFN. In porcine cells, G10 also activates the NF-κB signaling pathway, which is a priming signal for NLRP3 inflammasome activation. G10 induces potassium efflux and triggers NLRP3 inflammasome activation, which negatively regulates type I IFN.

Article Snippet: G10 (HY-19711), LPS (HY-D1056), ATP (HY-B2176), nigericin (HY-127019), MCC950 (HY-12815), and VX765 (HY-13205) were from MedChemExpress; and CL097 (tlrl-c97) was from InvivoGen.

Techniques: Activation Assay

Journal: Biomaterials Research

Article Title: Hypochlorous Acid-Responsive Prodrug Nanoplatform for Synergistic Cancer Immunotherapy

doi: 10.34133/bmr.0300

Figure Lengend Snippet: Schematic illustration of the design and mechanism of MD1a NP for synergistic cancer immunotherapy. (A) The HOCl-responsive methylene blue (MB)–doxorubicin (DOX) dimer prodrug co-assembles with the stimulator of interferon genes (STING) agonist 1a to form a tumor-activatable nanoplatform (MD1a NP). HOCl stimulation activates and triggers the release of MB and DOX, which in turn promotes nanoparticle disassembly and facilitates the subsequent liberation of 1a. (B) Following intravenous administration, elevated intratumoral HOCl triggers the activation and the subsequent release of MB and DOX, enabling synergistic photochemotherapy under near-infrared (NIR) laser irradiation. This process induces robust immunogenic cell death (ICD) while minimizing systemic off-target effects. Concurrently, HOCl-triggered nanoparticle disassembly accelerates 1a release, activating the STING pathway and establishing an immune-promoting tumor microenvironment (TME). In orthotopic 4T1 breast cancer mouse models, MD1a NP-mediated in situ tumor vaccination (ISTV) elicited strong antitumor immunity, effectively inhibiting both primary and distant tumor growth, preventing lung metastasis, and prolonging overall survival. PDT, photodynamic therapy; DAMPs, damage-associated molecular patterns; DC, dendritic cell.

Article Snippet: All solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. STING agonist 1a (HY-131994) and Cell Counting Kit-8 (CCK-8) were purchased from MedChemExpress, China.

Techniques: Activation Assay, Irradiation, In Situ