Journal: Frontiers in Immunology

Article Title: Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity

doi: 10.3389/fimmu.2017.01542

Figure Lengend Snippet: Induction of pro-inflammatory cytokines and activation of type-I interferon (IFN) by Panax notoginseng root (PNR) in vitro . (A–C) RAW 264.7 cells were treated with DMEM containing 10% fetal bovine serum (FBS) alone, 1,000 U/mL recombinant mouse IFN-β, or 100 µg/mL PNR and incubated at 37°C and 5% CO 2 . Supernatant from each group was harvested at 0 and 24 h and clarified by centrifugation at 2,500 g for 10 min at 4°C. Clarified supernatants were dispensed into murine interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IFN-β capture antibody-coated enzyme-linked immunosorbent assay plates to measure cytokine secretion. The test was performed in duplicate. (D) Western blotting was performed using the whole-cell lysates of macrophage-type cells treated with or without PNR (1, 10, and 100 µg/mL) to assess the expression of the nonphosphorylated and phosphorylated forms of IRF3, TANK-binding kinase 1 (TBK1), STAT1, and β-actin over time. Similar results were obtained, and the experiment was performed three times independently. Bar graph (mean ± SEM) statistics were determined using two-way ANOVA with Bonferroni’s correction (posttest), *** P < 0.001; ** P < 0.01; * P < 0.05. n.s., not significant.

Article Snippet: Anti-IRF3, anti-STAT1, anti-TBK1, anti-phospho-IRF3, anti-phospho-STAT1, and anti-phospho-TBK1 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), and antibodies targeting influenza proteins (NP, PA, HA, PB1, PB2, M1, M2, and NA) were obtained from GeneTex (San Antonio, TX, USA).

Techniques: Activation Assay, In Vitro, Recombinant, Incubation, Centrifugation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity

doi: 10.3389/fimmu.2017.01542

Figure Lengend Snippet: A schematic of the mechanism of Panax notoginseng root (PNR) in its enhancing of antiviral interferon-mediated immune responses and natural killer (NK) cell activity. PNR stimulates an antiviral state in murine macrophage cells by modulating the interferon (IFN) signaling pathway. PNR leads to the activation of IRF3 through TANK-binding kinase 1 (TBK1) and the production of type-I IFN. This secreted IFN can induce the inhibition of influenza A virus infection by stimulating the expression of stimulated target genes through the activation of signal transducer and activator of transcription 1, and these effects may be attributable to the ability of PNR to induce NK cell activity.

Article Snippet: Anti-IRF3, anti-STAT1, anti-TBK1, anti-phospho-IRF3, anti-phospho-STAT1, and anti-phospho-TBK1 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), and antibodies targeting influenza proteins (NP, PA, HA, PB1, PB2, M1, M2, and NA) were obtained from GeneTex (San Antonio, TX, USA).

Techniques: Activity Assay, Activation Assay, Binding Assay, Inhibition, Infection, Expressing

Journal: PLoS ONE

Article Title: Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

doi: 10.1371/journal.pone.0008734

Figure Lengend Snippet: MDDC were either untreated (none) or treated with BpWT or BpCyaA−. Phosphorylation of IRF3 was determined at the indicated time-points by Western blot. Data are from one representative out of three independent experiments performed with MDDC obtained from different donors.

Article Snippet: Rabbit polyclonal IgG anti-p-p44/42 mitogen-activated protein kinases (MAPK) (Thr202/Tyr204, ERK1/2), anti-p-p38 MAPK (Thr180/Tyr182), anti-p-JNK/Stress Activated Protein Kinase (SAPK) (Thr183/Tyr185), anti-p-I kappa B alpha (IκBα) (Ser32), anti-p-IRF3 Ab were from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Western Blot

Journal: Viruses

Article Title: THO Complex Subunit 7 Homolog Negatively Regulates Cellular Antiviral Response against RNA Viruses by Targeting TBK1

doi: 10.3390/v11020158

Figure Lengend Snippet: Overexpression of THOC7 negatively regulates the production of type I interferons (IFNs). ( A ) Interaction of THOC7 with TBK1 in a mammalian overexpression system. 293T cells were transfected with mock control, Flag-THOC7, and HA-TBK1 (10 μg each), followed by treatment with Sev or not for 10 h. At 24 h post-transfection, co-immunoprecipitation was performed with anti-HA beads and immunoblotting analysis with anti-Flag antibodies. ( B ) Small-scale screening of THOC7 functions in regulating SeV- or polyI:C-induced type I IFN signaling. 293T cells were seeded into 24-well plates and transfected with a luciferase reporter gene carrying the ISRE or IFN-β promoter (100 ng/well), pRL-TK (50 ng/well) and THOC7, followed by infection with SeV or transfection with polyI:C/TBK1 for 12 h. Relative luciferase activity levels were arbitrarily set to 0.1 (green). ( C – E ) THOC7 inhibited IFN-β promoter, ISRE, and NF-κB luciferase activation in a dose-dependent manner. Similar luciferase assay was performed, except with increasing amounts of the expression vector for THOC7. ( F ) THOC7 significantly reduced the phosphorylation and dimerization of IRF3. 293T cells were seeded into 6-well plates and transfected with HA-THOC7 (4 μg). After transfection for 12 h, the cells were treated with SeV or not for 10 h. Lysates were analyzed by native PAGE or SDS-PAGE. Quantification of western blotting bands from three independent experiments was performed with Image J software. ( G ) THOC7 inhibits IFN-β gene and ISG56 transcription. Similar transfection as described for ( F ) was performed. The mRNA levels were measured by q-PCR. Error bars indicate SD. n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant difference.

Article Snippet: Antibodies against the RLR signaling pathway components (sampler kit #8348), including IRF3, phosphorylated IRF3 (Ser396) (p-IRF3), and TBK1, were purchased from Cell Signaling Technology.

Techniques: Over Expression, Transfection, Control, Immunoprecipitation, Western Blot, Luciferase, Infection, Activity Assay, Activation Assay, Expressing, Plasmid Preparation, Phospho-proteomics, Clear Native PAGE, SDS Page, Software

Journal: Viruses

Article Title: THO Complex Subunit 7 Homolog Negatively Regulates Cellular Antiviral Response against RNA Viruses by Targeting TBK1

doi: 10.3390/v11020158

Figure Lengend Snippet: Knockdown of THOC7 leads to up-regulation of type I IFNs. ( A ) Immunoblot analysis following knockdown of transfected THOC7 in 293T cells transfected with THOC7-specific RNAis (2 μg), Flag-THOC7 plasmid (1 μg), and Flag-P50 (0.1 μg, as an internal control protein). ( B ) Real-time PCR of knockdown of exogenous THOC7 (left panel) or IFN-β mRNA (right panel) in 293T cells transfected with control RNAi or RNAis against THOC7 (4 μg). ( C ) Native PAGE analysis of IRF3 dimerization in 293T cells transfected with THOC7-specific RNAis (4 μg) and treated with SeV for 12 h. ( D ) Luciferase assays of 293T cells transfected with ISRE or IFNβ luciferase report plasmid (100 ng/well) with pRL-TK (50 ng/well), as well as THOC7 RNAi or control (0.5 μg/well). ( E ) IRF3 dimerization analysis of THOC7 RNAi#3 in gradient times of SeV treatment. Similar experiments were performed as ( C ) except with an increased time of SeV treatment. ( F ) Effects of knockdown of THOC7 on SeV-induced phosphorylation of IRF3. 293T cells were transfected with control RNAi or THOC7 RNAi#3 and infected with SeV for 10 h, followed by immunoblotting analysis with the indicated antibodies. Quantification of western blotting bands ( A , C , E , F ) from three independent experiments was performed with Image J software. Error bars indicate SD. n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant difference.

Article Snippet: Antibodies against the RLR signaling pathway components (sampler kit #8348), including IRF3, phosphorylated IRF3 (Ser396) (p-IRF3), and TBK1, were purchased from Cell Signaling Technology.

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Clear Native PAGE, Luciferase, Phospho-proteomics, Infection, Software

Journal: Viruses

Article Title: THO Complex Subunit 7 Homolog Negatively Regulates Cellular Antiviral Response against RNA Viruses by Targeting TBK1

doi: 10.3390/v11020158

Figure Lengend Snippet: THOC7 regulates the RLR signaling pathway by targeting TBK1. ( A ) A simulation model of node-activated pathway for the role of THOC7 in regulating the RLR signaling pathway. Downstream signaling was activated by transfection of the activated form of signaling pathway molecules containing RIG-I-N, MAVS, TBK1, IKKε, and IRF3-5D. ( B , C ) Effects of THOC7 overexpression or THOC7 knockdown on IFNβ or ISRE activation mediated by various activated forms of signaling molecules. 293T cells were seeded into 24-well plates and transfected with IFN-β or ISRE luciferase reporter (100 ng/well), pRL-TK (50 ng/well), THOC7 expression vector (0.5 μg, B), or THOC71 RNAi#3 constructs (0.5 μg, C) together with indicated activated forms of signaling molecules. Cells were harvested and analyzed by dual-luciferase reporter assay. After transfection for 24 h, the relative luciferase activities were normalized based on pRL-TK control activities. Error bars indicate SD. n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant difference.

Article Snippet: Antibodies against the RLR signaling pathway components (sampler kit #8348), including IRF3, phosphorylated IRF3 (Ser396) (p-IRF3), and TBK1, were purchased from Cell Signaling Technology.

Techniques: Transfection, Over Expression, Knockdown, Activation Assay, Luciferase, Expressing, Plasmid Preparation, Construct, Reporter Assay, Control