Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

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 ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; ****p<0.0001 by one-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 ± standard deviation is presented for n=3 independent experiments; **p < 0.01 and ***p < 0.001 by one-way ANOVA corrected by Dunnett’s test. H, HUVECs were treated as for panels A-C. Tissue factor mRNA levels were detected at 4 hours. Mean ± standard deviation is presented for n=3 independent experiments; ****p<0.0001 as compared by one-way ANOVA corrected by Dunnett’s test. I, HUVECs were treated as for panels A-C. Cell permeability was assessed by measuring horseradish peroxidase (HRP) movement through EC monolayers in a Transwell system as described in Methods. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; **p<0.01, ***p < 0.001 and ****p<0.0001 by two-way ANOVA corrected by Tukey’s test. # p<0.05, ### p<0.001, and #### p<0.0001 by two-way ANOVA corrected by Tukey’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

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

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

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 triggered by 10 μM calcium ionophore) for 4 hours. E-selectin (A), ICAM-1 (B), and VCAM-1 (C) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; ****p<0.0001 by one-way ANOVA corrected by Dunnett’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Isolation, Standard Deviation

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, NETs (1 μg DNA content/ml) were incubated with antibodies to histone H4 (100 ng/ml) for 1h, and then added to HUVECs for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C) and tissue factor (TF) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 one-way ANOVA corrected by Dunnett’s multiple comparison test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Incubation, Standard Deviation

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, HUVECs were pretreated with defibrotide (10 μg/ml) for 30 minutes, followed by recombinant histone H4 (25 μg/ml) for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C), and tissue factor (D) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; “***p < 0.001, ****p < 0.0001 by one-way ANOVA corrected by Dunnett’s test. E, Defibrotide, and histone H4 were incubated at 37°C for 30 minutes and then resolved on a 0.5% agarose gel. F, Surface plasmon resonance assay characterizing the binding kinetics of defibrotide to histone H4. The profile of defibrotide at gradient concentrations (from 0.39 μg/ml to 12.5 μg/ml) flowing over histone H4 protein immobilized on a NiNTA chip are shown. The calculated dissociation constant (K D ) is labeled.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Recombinant, Standard Deviation, Incubation, Agarose Gel Electrophoresis, SPR Assay, Binding Assay, Labeling

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, HUVECs were pretreated with defibrotide (10 μg/ml) for 30 minutes, followed by recombinant citrullinated histone H4 (25 μg/ml) for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C), and tissue factor (D) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA corrected by Dunnett’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Recombinant, Standard Deviation

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, HMVECs were pretreated with defibrotide (10 μg/ml) for 30 minutes, followed by recombinant histone H4 (25 μg/ml) for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C), and tissue factor (D) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA corrected by Dunnett’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Recombinant, Standard Deviation

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, HUVECs were pretreated with C29 (TLR2 inhibitor) or TAK242 (TLR4 inhibitor) for 1 hour, followed by the addition of histone H4 (25 μg/ml) for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C) and tissue factor (D) mRNA levels were determined by quantitative PCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA corrected by Dunnett’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Standard Deviation

Journal: medRxiv

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

doi: 10.1101/2021.02.21.21252160

Figure Lengend Snippet: A-D, HUVECs were pretreated with defibrotide (20 or 40 μg/ml) for 30 minutes, followed by TNF-α (20 nM) or LPS (1 µg/ml) for 4 hours. E-selectin (A), ICAM-1 (B), VCAM-1 (C), and tissue factor (D) mRNA levels were determined by qPCR. Mean ± standard deviation is presented for one representative experiment out of three independent experiments, all with similar results. *p<0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA corrected by Dunnett’s test.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Standard Deviation