Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A, B, E, and G) THP1-Lucia ISG (WT and STING KO) and RAW-Lucia ISG cells were treated with 2’3’-cGAMP (cGAMP, 0.5 μM) and/or indicated doses of MDAs or MMAE for 24 h, and the fold change of luminescence was normalized to DMSO or cGAMP-treated cells. (C, D, F, and H) THP1-Lucia ISG (WT and STING KO) and MEF cells were treated with cGAMP (0.5 μM) and/or indicated concentrations of MMAE for indicated times (C, D and F) or 6 h (H), and activation of the STING pathway were analyzed by immunoblotting. (I-L) THP1-Lucia ISG cells (WT and STING KO) and BMDMs (WT, Sting gt/gt , or Myd88 -/- mice) were stimulated with cGAMP and/or indicated concentrations of MMAE for 12 h (I, K) or 6 h (J, L). IFNβ production was measured by ELISA analysis (I and K). the activation of STING pathway was analyzed by immunoblotting (J). mRNA expression levels of IFNβ , CCL5 and CXCL10 in BMDMs (WT, Sting gt/gt , or Myd88 -/- mice) (n = 3 biological replicates) (L). cGAMP was used at 0.5 μM for all experiments unless otherwise noted.

Article Snippet: THP1-Lucia ISG cells (WT, STING KO) and BMDMs (WT, Sing gt/gt , Myd88 -/- mice) were seeded in 12-well culture plates, and treated with 0.5 μM cGAMP and/or MMAE (0.5 μM, 1 μM), or VcMMAE (0.5 μM) for 12 h. The concentration of IFNβ in cell supernatants or brain tissue homogenates were measured per the manufacturer’s instructions (Human/Murine IFN-beta bioluminescent ELISA kit, InvivoGen).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A) A diagram of MMAE promoting STING-mediated NF-kB signaling. (B-E) THP1-Lucia NF-kB (WT, p50 KO and p65 KO) and THP1-Lucia ISG (WT and p65 KO) were treated with cGAMP (0.5 μM) and/or indicated doses of MMAE for 24 h. The fold change of luminescence was normalized to DMSO-treated cells. (F, I and J) HEK293T cells were transiently transfected with STING plasmids (WT, Flag-hSTING (S366A)) for 24 hours. Cells were stimulated with cGAMP (0.5 μM) and/or MMAE (0.1 μM) for 6 h, and cell lysates were analyzed by immunoblotting for the indicated proteins (F and I). Quantification of LC3II/tubulin ratio from three independent experiments (J). (G) THP1-Lucai ISG (STING KO) cells stably expressing (hSTING WT, Flag-hSTING (S366A)) were stimulated with cGAMP and/or MMAE (1 μM) for 12 h. IFNβ production was measured by ELISA analysis. (H) STING was analyzed by immunoblotting in THP1-Lucai ISG (STING KO) cells.

Article Snippet: THP1-Lucia ISG cells (WT, STING KO) and BMDMs (WT, Sing gt/gt , Myd88 -/- mice) were seeded in 12-well culture plates, and treated with 0.5 μM cGAMP and/or MMAE (0.5 μM, 1 μM), or VcMMAE (0.5 μM) for 12 h. The concentration of IFNβ in cell supernatants or brain tissue homogenates were measured per the manufacturer’s instructions (Human/Murine IFN-beta bioluminescent ELISA kit, InvivoGen).

Techniques: Transfection, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A-L) THP1-Lucia ISG (WT and STING KO) cells were treated with cGAMP with or without indicated doses of MMAE for 24 h (A-F) or 6 h (J-L), and the fold change of luminescence was normalized to DMSO-treated cells. The activation of STING pathway was analyzed by immunoblotting (G-L). (M) THP1-Lucia ISG cells were pretreated for 12 h with or without anti-IFNAR2 antibody (20 μg/ml), and then stimulated with cGAMP or IFNβ (200 pg/ml) for 24 h in the absence or presence of MMAE (1 μM). Fold change of luminescence was normalized to DMSO-treated cells. (N and O) THP1-Lucia ISG (WT and IRF3 KO) cells were treated with cGAMP and/or indicated doses of MMAE for 12 h (N) or 6 h (O). IFNβ production was measured by ELISA analysis (N). Expression of IRF3 and the activation of STING pathway was analyzed by immunoblotting (N and O).

Article Snippet: THP1-Lucia ISG cells (WT, STING KO) and BMDMs (WT, Sing gt/gt , Myd88 -/- mice) were seeded in 12-well culture plates, and treated with 0.5 μM cGAMP and/or MMAE (0.5 μM, 1 μM), or VcMMAE (0.5 μM) for 12 h. The concentration of IFNβ in cell supernatants or brain tissue homogenates were measured per the manufacturer’s instructions (Human/Murine IFN-beta bioluminescent ELISA kit, InvivoGen).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A) Chemical structure of Monomethyl auristatin E (MMAE). (B) HeLa cells stably expressing human STING-GFP were stimulated with cGAMP (8 μM) and/or MMAE (1 μM), or VcMMAE (1 μM) for 2 h in the absence or presence of brefeldin A (BFA, 1 μM). Fluorescent images of cells were acquired on a Zeiss LSM980 Airyscan2 Confocal microscope using a 63× (NA 1.45) objective and processed in Zen Blue 3.1 software. STING (green), nuclei were stained with Hoechst (blue). Scale bars, 10 μm. (C-E) THP1-Lucia ISG cells were stimulated with cGAMP with or without MMAE (0.5 μM) or VcMMAE (0.5 μM) for 4 h (C and E). STING oligomerization was analyzed by native PAGE, and indicated proteins were detected by immunoblotting. The results are representative of three independent biological replicates (C). The activation of STING pathway was analyzed by immunoblotting (D). cGAMP quantification by LC-MS/MS in THP1-Lucia ISG cell lysates (E). (F) HeLa cells are stimulated similarly as in B. The STING (green) puncta are shown as 3D projections of Z-stack images. Scale bar, 5 μm. The STING puncta volume was quantitated by Imaris software (version 9.7) (n = 20). (G) THP1-Lucia ISG and RAW-Lucia ISG cells were treated with cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 24 h. Fold change of luminescence was normalized to DMSO-treated cells. (H and I) THP1-Lucia ISG cells were stimulated by cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 12 h (H), or 6 h (I). IFNβ induction was measured by ELISA and qPCR analysis.

Article Snippet: THP1-Lucia ISG cells (WT, STING KO) and BMDMs (WT, Sing gt/gt , Myd88 -/- mice) were seeded in 12-well culture plates, and treated with 0.5 μM cGAMP and/or MMAE (0.5 μM, 1 μM), or VcMMAE (0.5 μM) for 12 h. The concentration of IFNβ in cell supernatants or brain tissue homogenates were measured per the manufacturer’s instructions (Human/Murine IFN-beta bioluminescent ELISA kit, InvivoGen).

Techniques: Stable Transfection, Expressing, Microscopy, Software, Staining, Clear Native PAGE, Western Blot, Activation Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A and C-E) THP1-Lucia ISG cells (WT and STING KO) were infected with HSV-1-GFP (MOI = 1) or VSV-GFP (MOI = 0.1), and then cultured with cGAMP and/or MMAE (0.25 μM) for 24 h. The cells were then imaged with Olympus IX83 Inverted fluorescence microscope (A). The fluorescence intensity of virus-GFP was determined by ImageJ software, shown on the right of each row of images (n = 15, biological replicates). Scale bars, 100 μm. Cell viability was determined by ATP assay after indicated treatments (C). ISRE reporter activity was measured, and the fold change of luminescence was normalized to DMSO-treated cells (D and E). Bars are the means ± SEM. Significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. means non-significant. (B) THP1-Lucia ISG cells were infected with HSV-1-GFP (MOI = 1), and then cultured with cGAMP and/or MMAE (0.25 μM) with or without pretreatment of HSV-1-GFP (MOI = 1) or anti-IFNAR2 antibody (20 μg/ml) for indicated times. Expression of viral gene was determined by immunoblotting. (F) HeLa (STING deficient) and HeLa hSTING cells stably expressing human mCherry-TUBA1B were infected with HSV-1-GFP (MOI = 1), and then cultured with or without MMAE (0.1 μM) for 18–24 h. Representative confocal images of virus transport along microtubules were shown. Enlarged insets highlighted the co-localization of the viral particles (dashed white boxes) with intracellular microtubules. mCherry-TUBA1B (red), HSV-1-GFP (green). Scale bars, 10 μm. (G) Schematic diagram of microtubule-based transport of virus entry, replication, assembly, and egress from the host cell. MMAE-mediated microtubule network disruption seriously affects every process of viral replication and reproduction.

Article Snippet: THP1-Lucia ISG cells (WT, STING KO) and BMDMs (WT, Sing gt/gt , Myd88 -/- mice) were seeded in 12-well culture plates, and treated with 0.5 μM cGAMP and/or MMAE (0.5 μM, 1 μM), or VcMMAE (0.5 μM) for 12 h. The concentration of IFNβ in cell supernatants or brain tissue homogenates were measured per the manufacturer’s instructions (Human/Murine IFN-beta bioluminescent ELISA kit, InvivoGen).

Techniques: Infection, Cell Culture, Fluorescence, Microscopy, Virus, Software, ATP Assay, Activity Assay, Expressing, Western Blot, Stable Transfection, Disruption

Journal: STAR Protocols

Article Title: Protocol for the identification and expression analysis of a cytoplasmic membrane-localized protein STING

doi: 10.1016/j.xpro.2023.102172

Figure Lengend Snippet:

Article Snippet: Mouse: B16-Blue™ ISG-KO-STING , Invivogen , Cat#bb-kostg.

Techniques: Recombinant, Lysis, Membrane, Transfection, Cell Culture, Plasmid Preparation, Software, Microscopy

Journal: Med (New York, N.y.)

Article Title: Intestinal barrier dysfunction plays an integral role in arthritis pathology and can be targeted to ameliorate disease

doi: 10.1016/j.medj.2021.04.013

Figure Lengend Snippet:

Article Snippet: LIVE/DEAD™ Fixable Blue , Invivogen , Cat# L34961.

Techniques: Recombinant, Methylation, Isolation, SYBR Green Assay, Purification, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Software, Lysis, Plasmid Preparation