Journal: Frontiers in Immunology

Article Title: Analysis of Porcine RIG-I Like Receptors Revealed the Positive Regulation of RIG-I and MDA5 by LGP2

doi: 10.3389/fimmu.2021.609543

Figure Lengend Snippet: The signal activity of pLGP2, pRIG-I and pMDA5 before and after poly I:C transfection and virus infection. (A) 293T cells grown in 24-well plates (3×10 5 cells/well) were transfected with HA tagged pLGP2, pRIG-I, pMDA5 or pcDNA (0.5 μg each) using Lipofectamine 2000. Twenty-four hours post transfection, the cells were transfected with LMW poly I:C or HMW poly I:C (1 μg/ml) for 8h stimulation. The harvested cells were analyzed by RT-qPCR for downstream gene expressions as indicated. (B) 293T cells in 24-well plates (3×10 5 cells/well) were transfected as in (A) and infected with 0.01 MOI EMCV or VSV for 8 h, and analyzed by RT-qPCR. * p < 0.05, ** p < 0.01 vs pcDNA controls. # p < 0.05, ## p < 0.01 vs NS controls.

Article Snippet: Poly (I:C)-LMW, poly (I:C)-HMW, and poly (I:C) HMW Biotin were purchased from InvivoGen (San Diego, California, USA).

Techniques: Activity Assay, Transfection, Virus, Infection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Analysis of Porcine RIG-I Like Receptors Revealed the Positive Regulation of RIG-I and MDA5 by LGP2

doi: 10.3389/fimmu.2021.609543

Figure Lengend Snippet: Expressions of cellular genes and viral replications in LGP2, RIG-I and MDA5 homozygous KO PAM cell clones. (A) The LGP2 -/- , RIG-I -/- and MDA5 -/- PAMs plus CRISPR control PAMs grown in 12-well plates (5×10 5 cells/well) were transfected with LMW poly I:C or HMW poly I:C (1 μg/ml) for 8h. The expression of downstream cellular genes were analyzed by RT-qPCR. (B) The LGP2 -/- , RIG-I -/- and MDA5 -/- PAMs plus CRISPR control PAMs grown in 12-well plates (5×10 5 cells/well) were infected with EMCV at 0.01 MOI for 8 h. The cells were detected by Western-blotting with the indicated antibodies. (C, D) The LGP2 -/- , RIG-I -/- and MDA5 -/- PAMs plus CRISPR control PAMs in 12-well plates (5×10 5 cells/well) were infected with various viruses at MOI of 0.01 for 8-12 h and analyzed by RT-qPCR for the expressions of downstream cellular genes (C) and viral genes (up panel, D ). The supernatants from VSV and HSV1 infected PAMs were subjected to infection of Vero cells and the viral plaques were visualized at 30 h and 72 h post infection, respectively. The countable plaque numbers are labeled in red color below the wells of cell plates (low panel, D ). (E, F) MDA5 -/- PAMs (E) and RIG-I -/- PAMs (F) in 24-well plates were transfected different amounts of LGP2 and stimulated with 0.01 MOI VSV for 8 h. The downstream IFNβ and ISG56 were analyzed by RT-qPCR. ** p < 0.01 vs NS controls. # p < 0.05, ## p < 0.01 vs CRISPR controls in (A–D) , or pcDNA VSV controls in (E, F) .

Article Snippet: Poly (I:C)-LMW, poly (I:C)-HMW, and poly (I:C) HMW Biotin were purchased from InvivoGen (San Diego, California, USA).

Techniques: Clone Assay, CRISPR, Control, Transfection, Expressing, Quantitative RT-PCR, Infection, Western Blot, Labeling

Journal: Frontiers in Immunology

Article Title: Analysis of Porcine RIG-I Like Receptors Revealed the Positive Regulation of RIG-I and MDA5 by LGP2

doi: 10.3389/fimmu.2021.609543

Figure Lengend Snippet: Porcine LGP2 interacts RIG-I and MDA5 upon activation and promotes their RNA binding. (A) pLGP2-FLAG (1 μg) and pRIG-I-HA (1.5 μg) were co-transfected into 293T cells in 6-well plate (8×10 5 cells/well) for 24 h, then infected with or without 0.01 MOI EMCV for 8 h. The cells lysates were immunoprecipitated with anti-FLAG mAb and subjected to Western-blotting using the indicated antibodies. (B) pLGP2-FLAG and pMDA5-HA were co-transfected into 293T, and the transfected cells were treated and processed as in (A) . (C) pMDA5-FLAG and pRIG-I-HA were co-transfected into 293T cells, and the transfected cells were treated and processed as in (A) . (D) PAMs grown on 15-mm glass bottom cell culture dish (5×10 5 cells) were co-transfected with pLGP2-HA (0.75 μg) and pRIG-I-FLAG (0.75 μg) for 24 h, then infected with or without EMCV for 8 h. The cells were examined for co-localization by con-focal fluorescence microscopy. (E) pLGP2-HA and pMDA5-FLAG were co-transfected and the transfected PAMs were treated and examined for co-localization as in (D) . (F) pRIG-HA and pMDA5-FLAG were co-transfected, and transfected PAMs were treated and examined for co-localization as in (D) . (G) The purified pLGP2-HA, pLGP2-FLAG, pRIG-HA and pMDA5-FLAG were analyzed by SDS-PAGE. (H) pLGP2-HA, pRIG-I-HA and pMDA5-FLAG were incubated in tubes alone or together as indicated, then 1μg dsRNA poly (I:C) HMW Biotin and 30 μL streptavidin agarose were added for binding. The dsRNA bound proteins were analyzed by Western-blotting.

Article Snippet: Poly (I:C)-LMW, poly (I:C)-HMW, and poly (I:C) HMW Biotin were purchased from InvivoGen (San Diego, California, USA).

Techniques: Activation Assay, RNA Binding Assay, Transfection, Infection, Immunoprecipitation, Western Blot, Glass, Cell Culture, Fluorescence, Microscopy, Purification, SDS Page, Incubation, Binding Assay, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Cell reports

Article Title: Coordinated Viral Control by Cytotoxic Lymphocytes Ensures Optimal Adaptive NK Cell Responses

doi: 10.1016/j.celrep.2020.108186

Figure Lengend Snippet: (A) WT mice treated with αCD8-depleting antibody or isotype control antibody were challenged with MCMV, and viral titers were quantified from peripheral blood at day 6 PI. Data are pooled from two independent experiments with four or five mice per group. (B) Rag2 −/− mice were challenged with MCMV and treated daily with the antiviral drug ganciclovir or vehicle control starting from day 3 PI. The number of endogenous Ly49H + and Ly49H − NK cells was quantified in peripheral blood at day 7 PI. Data are representative of two independent experiments with seven or eight mice per group. (C) WT mice treated with αCD8-depleting antibody or isotype control antibody were challenged with MCMV. αCD8-treated mice were treated daily with ganciclovir or vehicle control starting from day 3 PI. The number of endogenous Ly49H + and Ly49H − NK cells was quantified in peripheral blood at day 7 PI. Data are pooled from three independent experiments with three to five mice per group. (D) WT mice were treated with a regular viral dose (Medium) or a 5- to 10-fold higher (High) or lower (Low) dose, and the number of endogenous Ly49H + and Ly49H − NK cells was quantified in peripheral blood at day 6 PI. Data are pooled from three independent experiments with four or five mice per group. (E) WT mice were challenged with MCMV and treated daily with the TLR3 agonist poly I:C or vehicle control starting from day 4 PI. The number of endogenous Ly49H + and Ly49H − NK cells was quantified in peripheral blood at day 7 PI. Data are representative of two independent experiments with three to five mice per group. Graphs present individual data points as scatter dot plots, and error bars show mean ± SEM. Statistical differences were calculated with an unpaired t test (A) and with a two-way ANOVA (B–E). A p value of <0.05 was used as the significance cut-off and is indicated with a single asterisk (*). See also .

Article Snippet: poly(I:C) (HMW) VacciGrade , InvivoGen , Cat#vac-pic.

Techniques: Control

Journal: Cell reports

Article Title: Coordinated Viral Control by Cytotoxic Lymphocytes Ensures Optimal Adaptive NK Cell Responses

doi: 10.1016/j.celrep.2020.108186

Figure Lengend Snippet:

Article Snippet: poly(I:C) (HMW) VacciGrade , InvivoGen , Cat#vac-pic.

Techniques: Control, Virus, Recombinant, Staining, SYBR Green Assay, Enzyme-linked Immunosorbent Assay

Journal: Microbiology and immunology

Article Title: Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex.

doi: 10.1111/j.1348-0421.2006.tb03865.x

Figure Lengend Snippet: Fig. 1. Phylogenetic tree of the 16S rRNA gene sequences of the three isolates CST 7247T, CST 7052, and CST 0506, and 52 slowly growing mycobacteria prepared by using the neighbor-joining method. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node. The tree was rooted with the use of Nocardia asteroides as the outgroup. The scale bar represents a 1% difference in nucleotide sequences.

Article Snippet: The 16S rRNA gene sequencing of strain CST 7247T (1,401 bp) was 98.6% (20 base mismatches) similar to M. terrae ATCC 15755T, whereas the 16S rRNA gene sequences of strains CST 7052 (1,442 bp) and CST 0506 (1,432 bp) were 99.9% (1 base mismatch) and 99.8% (3 base mismatches) similar, respectively, to M. arupense AR 3009T.

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms

doi: 10.4049/jimmunol.1801008

Figure Lengend Snippet: Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7−/−, TLR3−/−, MyD88−/−, Trif−/−) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. (A and B) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. (C and D) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. ***p < 0.001, #p < 0.0001, versus PBS group.

Article Snippet: Bone marrow–derived Mϕs (BMDM) were incubated in serum-free culture medium containing 0.05% BSA for 1 h before treatment with EVs (20 μg/ml), poly(I:C) (10 μg/ml; Enzo Life Sciences, Farmingdale, NY), R837 (1μg/ml; InvivoGen, San Diego, CA), or Pam3Cys (P3C; 1μg/ml; Enzo Life Sciences) for overnight.

Techniques: Isolation, Genetically Modified, Enzyme-linked Immunosorbent Assay