Journal: Scientific Reports

Article Title: Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene

doi: 10.1038/srep08696

Figure Lengend Snippet: (A) THP1-CD14 cells were infected with cell-free rBDV P/M-GFP at a multiplicity of infection of 0.1, and the resulting supernatants were collected. The SEAP activity was determined at the indicated time points (N = 3). (B) THP1-CD14 cells infected with rBDV P/M-GFP were stimulated with 100 ng/ml of TLR1/2 ligand (Pam3CSK4). At 48 h post-stimulation, GFP-positive cells were measured using an image-based cytometer. Error bars represent standard deviation of the mean (N = 3). **: P = 0.00474 (one-tailed t-test) (C) THP1-CD14 cells infected with rBDV P/M-GFP were stimulated with 100 ng/ml of TLR1/2 ligand (Pam3CSK4). The SEAP activities of the supernatants were determined at 24 h post-stimulation. Error bars represent the standard deviation of the mean (N = 3). **: P = 0.00761 (one-tailed t-test)

Article Snippet: To evaluate the inhibitory effects of the virus peptide on NF-κB activation, 2 × 10 5 THP1-CD14 cells were incubated with 100 μg/ml of each peptide for 4 h at 37°C, then subjected to stimulation with 100 ng/ml of Pam3CSK4, 10 ng/ml of TLR4 ligand (LPS), 10 ng/ml of TLR 2/6 ligand (FSL-1), 10 7 cells/ml of TLR2 ligand (heat killed L. Monocytogenes ), and 5 μg/ml of TLR 7/8 ligand (CL075) (all from InvivoGen), respectively.

Techniques: Infection, Activity Assay, Cytometry, Standard Deviation, One-tailed Test

Journal: Scientific Reports

Article Title: Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene

doi: 10.1038/srep08696

Figure Lengend Snippet: (A) Schematic diagram of NF-κB1 and BDV-N. (B) The crystallographic structure of the BDV-N tetramer and monomer (Protein Databank # 1N93). Yellow lines indicate the IPBN. (C) Inhibitory effect of the viral peptide on NF-κB activation. THP1-CD14 cells were pre-treated with 100 μg/ml of the viral peptide derived from BDV-N, a negative control peptide (CP7), or the positive control peptide (VIPER) and stimulated with five TLR ligands. At 24 h post-stimulation, the SEAP activities in the supernatants were measured. Error bars represent the standard deviation of the mean (N = 3).

Article Snippet: To evaluate the inhibitory effects of the virus peptide on NF-κB activation, 2 × 10 5 THP1-CD14 cells were incubated with 100 μg/ml of each peptide for 4 h at 37°C, then subjected to stimulation with 100 ng/ml of Pam3CSK4, 10 ng/ml of TLR4 ligand (LPS), 10 ng/ml of TLR 2/6 ligand (FSL-1), 10 7 cells/ml of TLR2 ligand (heat killed L. Monocytogenes ), and 5 μg/ml of TLR 7/8 ligand (CL075) (all from InvivoGen), respectively.

Techniques: Activation Assay, Derivative Assay, Negative Control, Positive Control, Standard Deviation

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: TLR4 gene and protein expression is upregulated in early skin wounds. (a) Microarray analysis shows that all four TLR4 probes in the array demonstrate a significant increase in expression, especially from 12h to 1 day (p<1.2×10 -7 , one-way ANOVA). (b) Real time PCR confirms that TLR4 mRNA is markedly increased in early stage wounds in TLR4 wild type mice (p=0.0003, one-way ANOVA). N=3 at each time point. (c) Photomicrographs of indirect immunofluorescence detection of TLR4 in skin wounds of TLR4 deficient and wild type mice. The control (rabbit IgG staining) histologic sections of wounds from TLR4 deficient mice, as well as the the 6h wound sample from wild type mice, were also stained with DAPI (blue). White arrows indicate wound edges; red arrow indicates fibrin clot, yellow arrow indicates advancing epidermal tip, and blue arrows show the higher magnification of the outlined areas. Bar=200μm.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: Mice with a TLR4 mutation exhibit impaired early wound healing without vascularity changes. (a) Representative photomicrographs of wounds at 0 and 6h as well as 1, 3, 5, 7 and 10 days after injury. Bar=3mm. (b) Percent of original wound size, N=5, * p<0.01, # p<0.05 VS wild type. (c) Photomicrographs of HE stained histologic sections of wounds. Bar=500μm. (d) Rate of wound re-epithelialization measured by histomorphometric analysis of tissue sections. N=5, * p<0.01 VS wild type. (e) Photomicrograpahs of ki67+ (red) proliferating keratinocytes with nuclear DAPI counterstaining (blue). Arrows indicate wound edges. Bar=200μm. (f) Percent area occupied by CD31 stained vessels at day 7 and 10 after wounding. N=5 at each time point. (g) Photomicrographs of Masson's trichrome stained histologic sections of day 10 wounds. Bar=500μm.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Mutagenesis, Staining

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: Wounds of TLR4 deficient and wild type mice show differences in inflammatory cell and cytokine content. (a, b, and c) The number of neutrophils, macrophages, and CD3+ T cells, respectively, in the wounds, determined by immunohistochemistry. (d, e, f, and h) mRNA levels of IL1β, IL-6, TNF-α and TLR2 in wounds, as determined by real time PCR. * p<0.01, # P<0.05, N=5 at each time point. Expression in the normal skin of wild type mice was used as baseline. (g) mRNA levels of IL1β, IL-6, TNF-α and EGF in epithelial cells at the 6h wound edges determined by LCM and RT-PCR. Data are averages of triplicate wells, and are representative of two independent experiments. * p<0.01 VS wild type.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: Scratch wound injury induces mRNA expression of TLR4 and production of inflammatory cytokines in NHEK cells. (a) LPS and injury-induced expression of TLR4 mRNA. # p<0.01 VS 1 and 3h expression in injured cells, and 8 and 24h LPS treated cells. THP1 treated with or without LPS were used as positive control. * p<0.0001 VS wounded and LPS treated NHEK. (b) Increased IL-1β and TNF-α mRNA expression in NHEK from 3 to 24h after wounding. # p<0.05 VS unwounded (IL-1β), * p<0.01 VS unwounded, 3h, and 8h (IL-1β), * p<0.01 and # p<0.05 VS unwounded (TNF-α), + p<0.01 VS 1h and 8h. Data in a&b represent the average of triplicate wells and are representative of two independent experiments. (c) Protein levels of IL-1β and TNF-α in the culture media 24h after injury. N=3, *p<0.01 VS unwounded cells. UW, unwounded; UT, LPS untreated.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Expressing, Positive Control

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: Scratch wound injury induces expression of p-p38 and p-JNK MAPK, but not NF-κB nuclear translocation in NHEK. Nuclear NF-κB, p-p38, and p-JNK were detected using immunofluorescence 15 min and 6h after wounding. To test the effects of anti-TLR4 antibody on induction of p-p38 (red) and p-JNK (green), NHEK were treated with anti-TLR4 or mouse IgG prior to scratch injury. (a, g, and m) Unwounded NHEK; (b, h, and n) 15 min after injury; (c, d, e, i, j, and k) 6h after injury; (p) TNF-α treated NHEK as NF-κB positive control; (f and l) Anisomycin treated NHEK as positive control for p-p38 and p-JNK; (a, d, g, h, j, and m) were also stained by DAPI (blue). UW: unwounded. Arrows indicate wound edges. Bar=100μm.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Expressing, Translocation Assay, Immunofluorescence, Positive Control, Staining

Journal: The Journal of investigative dermatology

Article Title: Toll-like receptor 4 plays an essential role in early skin wound healing

doi: 10.1038/jid.2012.267

Figure Lengend Snippet: Neutralization of TLR4 delays NHEK migration and abolishes IL-1β production in injured NHEK. (a and b) NHEK monolayers were pretreated with anti-TLR4 or normal mouse IgG (20μg/ml) followed by mitomycin C (10μg/ml) treatment, and then wounded by scratches. The defined areas were photographed at 0 and 16h after wounding. The numbers of cells migrated out of the initial areas were counted. N=4. * p<0.05 compared to IgG control. (c and d) Cultured NHEK were incubated with anti-TLR4 and/or p38 inhibitor SB203580, and/or JNK inhibitor SP600125 and then cells were injured by scratching. The supernatants were collected 24h later for IL-1β and TNF-α ELISA analyses. N=3,*p<0.01, # p<0.05 compared to wounded untreated cells. UW, unwounded. Bar=100μm.

Article Snippet: To examine the role of TLR4 and its signaling pathway in the production of IL1β and TNF-α, cells were incubated with anti-TLR4 antibody (20μg/ml) and/or p38 inhibitor SB203580 (5μM), and/or JNK inhibitor SP600125 (0.15μM) (both from LC Laboratories, Woburn, MA) for 90 min prior to injury.

Techniques: Neutralization, Migration, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: S100A8-Mediated NLRP3 Inflammasome-Dependent Pyroptosis in Macrophages Facilitates Liver Fibrosis Progression.

doi: 10.3390/cells11223579

Figure Lengend Snippet: Figure 5. TLR4/NF-κB signaling cascade and ROS abundance are responsible for S100A8-induced NLRP3 inflammasome-dependent pyroptotic death in macrophages. (A) Western blot analysis of p65, p-p65, IKKα, and p-IKKα expression in THP-1 macrophages treated with GST-rhS100A8 or GST for 0, 30, 60 or 120 min. The protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes relative to the 0 min group (right panel). (B–E) THP-1 macrophages

Article Snippet: To investigate the endogenous PRR of S100A8, THP-1 differentiated macrophages were pretreated with the TLR4 inhibitor TAK-242 (10 μM, S7455, Selleck, Houston, Texas, USA) or the receptor for advanced end products (RAGE) inhibitor FPS-ZM1 (10 μM, S8185, Selleck, Houston, Texas, USA) for 1 h and then stimulated with rhS100A8 (5 μg/mL).

Techniques: Western Blot, Expressing