Journal: Nature microbiology

Article Title: TRIM23 mediates virus-induced autophagy via activation of TBK1

doi: 10.1038/s41564-017-0017-2

Figure Lengend Snippet: a, Summary of the TRIM cDNA screen (shown in ) in which 61 TRIM proteins were tested for their ability to induce GFP-LC3B puncta formation in transiently transfected HeLa cells. TRIM proteins were categorized into ‘high inducers’ of autophagy (red; defined as inducing more GFP-LC3B puncta than rapamycin stimulation), or ‘low/intermediate inducers’ and ‘non-inducers’ (magenta/blue; defined as inducing fewer GFP-LC3B puncta than rapamycin but more than empty vector transfection, or similar to vector transfection, respectively). b, Representative laser-scanning confocal microscopy images of GFP-LC3B puncta formation in HeLa cells for the top 7 hits from the TRIM cDNA screen shown in (a). Representative images for GFP-LC3B puncta formation induced by rapamycin treatment or empty vector transfection (controls) are also shown. Scale bar, 20 µm. c–e, GFP-LC3B puncta formation in HeLa cells transiently transfected with non-targeting control siRNA (NT) or siRNAs targeting the indicated TRIM proteins and subsequently infected with mutHSV-1 (MOI 4) for 8 h (c), IAV (MOI 5) for 14 h (d), or EMCV (MOI 100) for 6 h (e). Box and whisker plots show the distribution of the area of GFP-LC3B for 3 individual images, each containing ~30 cells. f, Summary of the results of GFP-LC3B puncta formation from the RNAi screen using viral stimuli (c-e) or rapamycin treatment . NV, no infection. *p<0.05, ***p<0.0005 (ANOVA test). Data are representative of one screen ( a ), or two ( b ) or three ( c–f ) independent experiments.

Article Snippet: RSV (A2 strain) and EMCV (EMC strain) were purchased from ATCC.

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Control, Infection, Whisker Assay

Journal: Scientific Reports

Article Title: High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

doi: 10.1038/srep00367

Figure Lengend Snippet: The concentration of serum TNF-α was determined at 0, 2, and 8 h after LPS treatment in each EMCV infection period (A). TNF-α mRNA expression in the brain, heart, liver, lung, and spleen at 0, 1, and 8 h after LPS treatment in each EMCV infection period was determined on the basis of 18S rRNA expression using real-time RT-PCR. The data were calculated referring to mRNA levels of the respective tissues in control mice (0 days after EMCV infection, 0 hours after LPS inoculation) (B). The data are represented as means ± SD of the results of 4 mice in each group. * p <0.05

Article Snippet: A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan).

Techniques: Concentration Assay, Infection, Expressing, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

doi: 10.1038/srep00367

Figure Lengend Snippet: Histopathological examination in the brain and heart at 0, 2 and 5 days after EMCV infection was performed. Tissue sections were deparaffinized, stained with hematoxylin-eosin, and examined under light microscopy. Scale bars, 200 μm (low-power field) and 50 μm (high-power field). These experiments were performed with 4 mice in each group and produced the same results (A). Viral RNA in the brain, heart, liver, lung, and spleen at 0, 2, and 5 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data are represented as means ± SD of the results of 4 mice in each group. The statistical analysis was performed by comparing with Day 2 (B). * p <0.05

Article Snippet: A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan).

Techniques: Infection, Staining, Light Microscopy, Produced, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

doi: 10.1038/srep00367

Figure Lengend Snippet: The expression of MCP-1 (A), MIP-2 (B) and KC (C) mRNA in the brain, heart, liver, lung and spleen at 0, 2 and 5 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data were calculated referring to mRNA levels of the respective tissues in control mice (Day 0). The data are represented as means ± SD of the results of 6 mice in each group. * p <0.05

Article Snippet: A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

doi: 10.1038/srep00367

Figure Lengend Snippet: The expression TLR4 mRNA in the brain, heart, liver, lung, and spleen at 0, 2, and 5 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data were calculated referring to mRNA levels of the respective tissues in control mice (Day 0). The data are represented means ± SD from 4 mice of each group (A). Following reaction with anti-CD16/CD32 antibody to suppress nonspecific binding, MNCs from the heart were stained with anti-CD3ε, anti-CD11b, anti-CD11c, anti-CD19, anti-CD49b and anti-TLR4 antibody. The positive rate of the cells was made on the basis of isotype control. The data are representative of three separate experiments (B). * p <0.05

Article Snippet: A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Binding Assay, Staining

Journal: Scientific Reports

Article Title: High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

doi: 10.1038/srep00367

Figure Lengend Snippet: The mice were inoculated intravenously with brefeldin A (250 μg/mouse) and LPS (10 μg/mouse) at 5 days after EMCV infection, and MNCs from the heart were harvested at 1 h after LPS treatment. MNCs were stained with anti-CD11b and anti-TNF-α antibody. The data are representative of three separate experiments (A). Isolated CD11b + and CD11b − MNCs and total cells before isolation were cultured at 1×10 5 cells per 200 μL with LPS (1 μg/mL) for 24 h. The concentration of TNF-α was determined in culture supernatant. The data are represented means ± SD in triplicate cultures (B). * p <0.05

Article Snippet: A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan).

Techniques: Infection, Staining, Isolation, Cell Culture, Concentration Assay