Journal: Virology Journal

Article Title: Interleukin-17A pretreatment attenuates the anti-hepatitis B virus efficacy of interferon-alpha by reducing activation of the interferon-stimulated gene factor 3 transcriptional complex in hepatitis B virus-expressing HepG2 cells

doi: 10.1186/s12985-022-01753-x

Figure Lengend Snippet: Repression of IL-17A pathway by TRAF6 inhibitor reversed the inhibitory effect of IL-17A on IFN-α-induced ISGF3 activation and anti-HBV activity. HepG2-HBV1.3 cells were pre-incubated with or without 5 uM or 20 uM TRAF6 inhibitor C25-140 for 6 h, then cells were cultured for another 24 h in this medium with or without supplementary 50 ng/ml IL-17A, and followed by co-treatment with or without 1000 IU/ml IFN-α for 24 h. A Culture supernatants were collected for assay of levels of HBsAg and HBeAg by ELISA. Data were displayed as a percentage of the values obtained for mock-treated cells. B Cell lysates were prepared for detection of protein expression by western blotting. Relative expression of protein was shown as fold-change in comparison with the cells treated with IFN-α alone. C Total cellular RNA was extracted for detection of mRNA expression of anti-HBV ISGs (ISG15 and MxA) by RT-qPCR. Relative expression of mRNA was shown as fold-change compared to the mock-treated cells. All data were shown as mean ± standard deviation (SD) (error bars) from at least 3 independent experiments. p < 0.05 is considered statistically significant. *p < 0.05, **p < 0.01 between two indicated groups

Article Snippet: They were first incubated with C25-140 (5 or 20 µM) (MedChemExpress LLC, USA) for 6 h and then treated with IL-17A (50 ng/ml) (PeproTech, UK) for 24 h, followed by combined treatment with recombinant human IFN-α-2b (1000 IU/ml) (Anhui Anke Biotechnology, China) and IL-17A for another 24 h. The cell culture supernatant was collected to assay HBsAg and HBeAg levels.

Techniques: Activation Assay, Activity Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Comparison, Quantitative RT-PCR, Standard Deviation

Journal: JCI Insight

Article Title: Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide

doi: 10.1172/jci.insight.149149

Figure Lengend Snippet: ( A – C ) HUVECs were pretreated with defibrotide (10 μg/mL) for 30 minutes, followed by isolated NETs (1 μg DNA content/mL) for 4 hours. E-selectin ( A ), ICAM-1 ( B ), and VCAM-1 ( C ) mRNA levels were determined by qPCR. Mean ± SD is presented for 1 representative experiment out of 3 independent experiments, all with similar results; **** P < 0.0001 by 1-way ANOVA corrected by Dunnett’s test. ( D – F ) HUVECs were pretreated with defibrotide (10 μg/mL) for 30 minutes, followed by the addition of NETs for 6 hours. Surface expression of E-selectin ( D ), ICAM-1 ( E ), and VCAM-1 ( F ) were then detected by in-cell ELISA. ( G ) HUVEC monolayers were pretreated with defibrotide (10 μg/mL) for 30 minutes, followed by NETs (1 μg DNA content/mL) for 4 hours. Calcein-AM–labeled neutrophils were then added as described in Methods. Mean ± SD is presented for n = 3 independent experiments; ** P < 0.01 and *** P < 0.001 by 1-way ANOVA corrected by Dunnett’s test. ( H ) HUVECs were treated as for A – C . Tissue factor mRNA levels were detected at 4 hours. Mean ± SD is presented for 1 representative experiment out of 3 independent experiments, all with similar results; **** P < 0.0001 as compared by 1-way ANOVA corrected by Dunnett’s test. ( I ) HUVECs were treated as for A – C . Cell permeability was assessed by measuring horseradish peroxidase (HRP) movement through EC monolayers in a Transwell system as described in Methods. Mean ± SD is presented for 1 representative experiment out of 3 independent experiments, all with similar results; ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by 2-way ANOVA corrected by Tukey’s test. # P < 0.05, ### P < 0.001, and #### P < 0.0001 by 2-way ANOVA corrected by Tukey’s test.

Article Snippet: Soluble E-selectin and P-selectin were quantified in mice sera using the mouse E-selectin Duoset ELISA (DY575, R&D system) and mouse P-selectin Duoset ELISA (DY737, R&D system) according to the manufacturer’s instructions.

Techniques: Isolation, Expressing, In-Cell ELISA, Labeling, Permeability

Journal: JCI Insight

Article Title: Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide

doi: 10.1172/jci.insight.149149

Figure Lengend Snippet: ( A and B ) HUVECs were treated with histone H4 (100 μg/mL) ± defibrotide (20 μg/mL) for 4 hours. The concentrations of IL-1β ( A ) and IL-18 ( B ) were determined in supernatants ( n = 6 independent experiments); *** P < 0.001 and **** P < 0.0001 by 1-way ANOVA corrected by Dunnett’s test. Data were presented as mean ± SD. ( C ) Immunoblotting detection of activated gasdermin D (GSDMD) and caspase 3 in cell lysates. HUVECs were treated with histone H4 (100 μg/mL) or staurosporine (50 nM) for 6 hours before collecting the cell lysates. Con, control; H4, histone H4; stauro, staurosporine. ( D and E ) HUVECs were treated as in A and B , and HMGB1 translocation ( D ) and secretion ( E ) were determined by microscopy and supernatant ELISA, respectively ( n = 3 independent experiments); **** P < 0.0001 by 1-way ANOVA corrected by Dunnett’s test. Scale bars: 100 μm (primary image) and 10 μm (inset). Data were presented as mean ± SD.

Article Snippet: Soluble E-selectin and P-selectin were quantified in mice sera using the mouse E-selectin Duoset ELISA (DY575, R&D system) and mouse P-selectin Duoset ELISA (DY737, R&D system) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Translocation Assay, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide

doi: 10.1172/jci.insight.149149

Figure Lengend Snippet: ( A ) Thrombus initiation in the IVC via placement of a fixed suture over a spacer that was subsequently removed. ( B ) Mice were injected with either histone (10 mg/kg) or saline via tail vein 1 hour prior to surgery. Meanwhile, defibrotide (150 mg/kg) or saline was administered by retro-orbital injection 24 hours prior to surgery and then immediately following closure of the abdomen. Thrombus weight was determined 24 hours later. Scatter plots are presented, with each data point representing a unique mouse (horizontal bars represent mean + SD); * P < 0.05 and ** P < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparison test. Data were presented as mean ± SD. ( C ) Representative thrombi from the experiments presented in panel B with rulers measuring thrombi in millimeters. ( D and E ) Serum samples from the experiments presented in B were tested for soluble E-selectin ( D ) and soluble P-selectin ( E ) by ELISA; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by 1-way ANOVA corrected by Dunn’s multiple comparison test. Data were presented as mean ± SD. ( F – H ) Thrombus sections from B were stained for Ly6G + and CD45 + cells. Positively stained cells were quantified in 4 randomly selected fields for each thrombus. ** P < 0.01 and *** P < 0.001 by 1-way ANOVA corrected by Dunn’s multiple comparison test. Scale bars: 1000 μm. Data is presented as mean ± SD.

Article Snippet: Soluble E-selectin and P-selectin were quantified in mice sera using the mouse E-selectin Duoset ELISA (DY575, R&D system) and mouse P-selectin Duoset ELISA (DY737, R&D system) according to the manufacturer’s instructions.

Techniques: Injection, Saline, Comparison, Enzyme-linked Immunosorbent Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Functions of Small Organic Compounds that Mimic the HNK-1 Glycan

doi: 10.3390/ijms21197018

Figure Lengend Snippet: Small organic compounds inhibit the binding of the HNK1-specific antibody to the HNK1 mimetic peptide in a concentration-dependent manner. ( a ) HNK1-specific antibody was substrate-coated on an enzyme-linked immunosorbent assay (ELISA) plate and preincubated with different concentrations (1–200 μM) of compounds from libraries or the negative control compound tacrine. Biotinylated HNK1 mimetic peptide was then added and detected by horseradish peroxidase (HRP)-coupled streptavidin followed by development with o-phenylenediamine dihydrochloride (OPD) substrate to produce a colored product that was quantified in the ELISA reader. The signal with this peptide without compounds was set to 100%. The graph shows average relative inhibition of the signal (duplicate wells carried out in three independent experiments ± SEM). All data points below the asterisks are different in inhibition when compared to the negative control compound tacrine (one-way ANOVA, F(31/64) = 4.122, p < 0.0001; Fisher’s PLSD test, * p < 0.05, ** p < 0.01). ( b ) Structure of the HNK1 mimetic compounds.

Article Snippet: The secondary donkey anti-mouse IgM antibody coupled to horseradish peroxidase (HRP) (cat #715-035-020) and secondary donkey anti-rabbit IgG antibody coupled to alexa fluor ® 488 (cat #711-545-152) were from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Inhibition