Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Evaluation of a new commercial assay for the measurement of tissue factor activity of extracellular vesicles isolated from human plasma

doi: 10.1016/j.rpth.2025.103007

Figure Lengend Snippet: Measurement of extracellular vesicle (EV) tissue factor (TF) activity using the CY-QUANT MV-TF (CY) and Chapel Hill (CH) assays in the presence of an anti-TF pathway inhibitor (TFPI) antibody and levels of TFPI in EVs. Plasma was prepared from the whole blood of 4 healthy donors with lipopolysaccharide (+LPS) or without lipopolysaccharide (−LPS) stimulation. EVs were isolated from plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C. (A) TF-dependent activated factor X generation was measured using the CY (yellow) and CH (blue) assays in the presence of an inhibitory anti-TFPI antibody (2H8). (B) TFPI was analyzed in EV samples from −LPS and +LPS samples by Western blotting. A polyclonal rabbit anti-human TFPI antibody was used (final concentration 3 μg/mL) to detect TFPI. A goat anti-rabbit immunoglobulin G secondary antibody (Cell Signaling, Cat. number 7074) was used at a 1:5000 dilution to detect the anti-TFPI antibody. The position of TFPI and molecular weight (MW) standards is shown. (C) Bands were quantified using ImageLab software. The Wilcoxon matched-pairs signed-rank test was used to compare the 2 groups. Data are shown as mean ± SD or individual values. ∗∗ P < .01.

Article Snippet: The membranes were incubated with a rabbit polyclonal anti-TFPI antibody [ ] (final concentration 3 μg/mL in 5% bovine serum albumin in 1× Tris-buffered saline with 0.1% Tween-20) overnight at 4 °C with rocking, washed, and then incubated with a goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Cell Signaling, Cat. number 7074, 1:5000 in blocking buffer) for 1 hour at room temperature with rocking.

Techniques: Activity Assay, Clinical Proteomics, Isolation, Centrifugation, Western Blot, Concentration Assay, Molecular Weight, Software

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) PABA/NO (25 µM) addition to HL60 cells corresponds with an S-shaped generation of NO, becoming linear after cells are pretreated with 50 nM L-NAME for 30 min prior to PABA/NO (100 and 200 nM of L-NAME addition did not show any difference). DETA/NO and DEA NONOate (25 µM) addition to HL60 cells result in linear NO generation kinetics. B) Emission and excitation spectra of a purified fluorescent nitro-aromatic product (MW 622 Da) C) and D) Short and long exposure of HL60 cells to 25 µM PABA/NO produces linear kinetics for the fluorescent nitro-aromatic product (MW 622 Da) and S-shaped kinetics for NO generation. Data are: trace smoothed with Sigma Plot 10 software (panel A) and original traces (Panels B, C and D) representative of three independent experiments. Arrows indicate addition of drug.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Purification, Software

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) Fluorescent and DIC images of cells before (upper panels) and after (lower panels) transfection with FITC-labeled siRNA. B) Viability of mock (black column) and eNOS siRNA transfected cells (white column) detected by trypan blue exclusion. C) Immunoblot analysis of cell lysates (∼100 µg of total protein/lane) compared to standard (STD, Cayman) with eNOS detection. D) Immunoblot (panel C) quantification. E) Immunoblot analysis of eNOS in HL60 cells (1) and in HDMVEC (2) lysates (∼20 µg of total protein/lane); Data in B) and D) represent mean ± SE of 3 independent experiments. Loading controls with GAPDH detection on the same blot are presented.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Transfection, Labeling, Western Blot

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) HL60 and TReNOS −/− cells labeled with DAF-FM to measure total basal NO levels. B). NO generation in HL60 cells following PABA/NO, PABA, or ThG titration. C) Kinetics of intracellular Ca 2+ increase in TReNOS −/− after PABA/NO (25 µM) or ThG (20 nM) addition. D) Growth curves for HL60 and TReNOS −/− cells. Data represent: mean ± SE (panels A and B) for 3 independent experiments; representative traces of 3 independent experiments (panel C); and average ± variance for 2 independent experiments (panel D).

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Labeling, Titration

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) Basal NO levels in HL60 and TReNOS −/− cells before and after incubation with 50 nM of the specific CaM-inhibitor W-7. B) Kinetics of NO generation in HL60, HL60 cells after preincubation (30 min) with 50 nM of W-7, and in TReNOS −/− cells following PABA/NO (25 µM) addition. Data represent: mean ± SE (panel A) and representative traces (panel B) of 3 independent experiments.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: Estimated basal and PABA/NO-induced intracellular NO levels.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques:

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) Control and PABA/NO-treated (25 µM, 30 min) HL60 cell lysates (∼100 µg protein/lane) resolved by non-reducing SDS-PAGE and evaluated by immunoblot with eNOS polyclonal primary antibodies and PSSG monoclonal primary antibodies and detected simultaneously with both: anti-mouse (red) and anti-rabbit (green) fluorescent secondary antibodies. B) Fluorescent detection (TG-1) of cytosolic protein sulfhydryls in HDMVE and in HL60 cells before (CTR) and after: PABA/NO (20 µM, 30 min) or ThG (200 nM, 30 min) addition. C) Immunoprecipitation of eNOS from HDMVEC with S-glutathionylated protein antibody. The blot was developed with the eNOS antibodies. Lanes are: 1- control HDMVEC lysate; 2- PABA/NO-treated (20 µM, 30 min) cell lysate; 3- IP of control cell lysate; 4- IP of PABA/NO-treated cell lysate; 5 -IP of PABA/NO-treated cell lysate after TCEP treatment. Arrows indicate low and high molecular weight eNOS bands. Loading controls are actin immunostaining. Data are: representative blots (panels A, C) and mean ± SD of 3 independent experiments, statistical significance p≤0.001.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Control, SDS Page, Western Blot, Immunoprecipitation, High Molecular Weight, Immunostaining

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) Fluorescent detection (TG-1) of dose-dependent HL60 surface protein sulfhydryls level after PABA/NO, PABA and ThG addition. B). PABA (50 µM) addition to HL60 cells (with or without extracellular Ca 2+ ) did not initiate generation of fluorescent metabolite (MW 622). C) Plasma trans-membrane potential (V m ) recorded as Bis-Oxanol emission at 520 nm after addition of 20 nM ThG, 25 µM PABA or 25 µM PABA/NO in HL60 cells. D) DETA/NO and DEA NONOate (25 µM) addition to HL60 cells did not initiate Ca 2+ fluxes. Data represent: mean ± SE (panel A), representative traces averaged and smoothed with Sigma Plot 10.0 software (panel C), and original representative traces (panel B and D) of 3 independent experiments.

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: Clinical Proteomics, Membrane, Software

Journal: PLoS ONE

Article Title: Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

doi: 10.1371/journal.pone.0014151

Figure Lengend Snippet: A) Scheme of nitro-aromatic PABA/NO-GSH adduct (MW 622 Da) generation. B) HPLC analysis of PABA/NO-GSH reaction in vitro and detection of PABA standard under the same conditions. C) HPLC analysis of HL60 lysate after incubation with 50 µM PABA/NO (1) and after “spike” with the purified nitro-aromatic adduct (MW 622 Da, 2). D) ESI MS analysis of purified from in vitro reaction nitro-aromatic PABA/NO-GSH adduct (m/z 623.8, left panel) and HL60 cells lysate after their incubation with purified nitro-aromatic adduct (m/z 623.8) product (right panel, indicated with asterisk).

Article Snippet: The human promyelocytic leukemia cell line HL60 was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO 2 atmosphere at 37°C.

Techniques: In Vitro, Incubation, Purification

Journal: Scientific Reports

Article Title: A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

doi: 10.1038/s41598-021-89957-z

Figure Lengend Snippet: Assessment and characterization of the multimeric ACE2 proteins. The three ACE2 proteins used in this study were characterized for their molecular properties. 200 μg of ACE2-Fc, ACE2-Fc-TD or sACE2 were separated by SDS-PAGE alongside protein molecular weight markers as indicated ( A ). The molecular size of the ACE2 complexes determined by SEC–MALLS experiments. The molecular weight of the small peak in the ACE2-Fc-TD SEC–MALLS (indicated by the blue arrow, C ) was not calculated due to low signal and likely corresponds to a mixture of higher molecular weight species observed in the AUC data ( D , E ). The molecular sizes were corroborated using AUC determining sedimentation coefficient distributions for ACE2-Fc and ACE2-Fc-TD ( D , E ). Data are shown for three samples of each protein measured at different protein concentrations. The effect of ACE2 multimerization on SARS-CoV-2 binding affinity was established using SPR ( F – H ). Biotinylated SARS-CoV-2-RBD was captured on a streptavidin-coated chip and ACE2 antibodies flowed over the surface at 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM. Sensograms are shown for sACE2 ( F ), ACE2-Fc ( G ) and ACE2-Fc-TD ( H ). The dissociation rates of the multimeric species ACE2-Fc and ACE2-Fc-TD are slower than for monomeric sACE2, leading to higher affinity of ACE2-Fc-TD for SARS-Cov-2-RBD. Kinetic parameters and data fits are shown in Supplementary Fig. . Uncropped images, showing relevant parts full length Coomassie stained SDS-PAGE gels (with indicated stained molecular weight markers), are displayed in Supplementary Fig. . A ChemiDoc Imaging system (BioRad) was used to capture images.

Article Snippet: 60 μg of pTT5-CoV-2 RBD plasmid DNA was added to 3 mL of Opti-Mem (Thermofisher). and 160ul of ExpiFectamine (Thermofisher) added to 3 mL of Opti-Mem and both incubated for 5 min at room temperature.

Techniques: SDS Page, Molecular Weight, Sedimentation, Binding Assay, Staining, Imaging

Journal: Scientific Reports

Article Title: A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

doi: 10.1038/s41598-021-89957-z

Figure Lengend Snippet: ELISAs with ACE2 protein binding to SARS-CoV-2 spike protein. The ability of ACE2 Quads to bind to SARS-Cov-2 virus spike protein was analysed by binding assays with and without anti-SARS-Cov-2 tetramers. ( A ) Direct ELISAs of ACE2 Binding to SARS-Cov2-RBD. Increasing amounts of ACE2 proteins were added to SARS-CoV-2 RBD protein coated ELISA plates followed by detection with 100ul of anti- IgG-HRP and, after washing, TMB was added for 20 min and the absorbance read at 450 nm. The ACE2-Fc-TD EC 50 ~ 2 pM and ACE2-Fc EC 50 ~ 6 pM, representative of two experiments. ( B ) Sandwich ELISA detection of anti-SARS-CoV-2 spike protein. ACE2-IgG-TD coated ELISA wells (300 ng/well as the capture protein) were incubated with SARS-CoV-2 RBD in the range of 0-300 nM in PBS containing 0.05% Tween-20 overnight at 4 °C. Anti-RBD antibodies were added at 1 nM in PBS containing 0.05% Tween-20 and incubated for 3 h at room temperature and bound antibody detected with anti-His-HRP and, after washing, addition of TMB and the absorbance read at 450 nm. ( C ) Direct ELISAs of anti-CoV-2 spike protein binding antibodies. ELISA wells were coated with 200 ng SARS-CoV-2 RBD protein 200 ng/well) in PBS, pH 7.4 and detected with the range of anti-CoV-2 antibodies. The bound antibody was detected with an HRP-conjugated anti-His antibody followed by addition of TMB and determining the absorbance read at 450 nm.

Article Snippet: 60 μg of pTT5-CoV-2 RBD plasmid DNA was added to 3 mL of Opti-Mem (Thermofisher). and 160ul of ExpiFectamine (Thermofisher) added to 3 mL of Opti-Mem and both incubated for 5 min at room temperature.

Techniques: Protein Binding, Binding Assay, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Incubation

Journal: Scientific Reports

Article Title: A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

doi: 10.1038/s41598-021-89957-z

Figure Lengend Snippet: Tetrameric ACE2-Fc-TD is a potent inhibitor of virus infection. The viral infection neutralizing potency of tetrameric ACE2-Fc-TD Quad was examined in SARS-CoV-2 pseudovirus infection assays. Lentiviral pseudotyped virus expressing the SARS-CoV-2 spike protein was incubated for 1 h with SAD-S35 (AcroBiosystems) and ACE2-Fc-TD alongside either tetrameric scFv or Fab variants of CR3022. HEK 293 T/17 cells expressing ACE-2 and TMPRSS2 were subsequently incubated with the mixtures and luciferase activity detected after 60 h. Graphs represent the percentage neutralization plotted as μg/mL ( A ) or molarity ( B ) calculated by non-linear regression analysis. Data are represented as mean ± SEM. IC 50 values shown in ( C ) were calculated using GraphPad Prism software.

Article Snippet: 60 μg of pTT5-CoV-2 RBD plasmid DNA was added to 3 mL of Opti-Mem (Thermofisher). and 160ul of ExpiFectamine (Thermofisher) added to 3 mL of Opti-Mem and both incubated for 5 min at room temperature.

Techniques: Infection, Expressing, Incubation, Luciferase, Activity Assay, Neutralization, Software

Journal: Scientific Reports

Article Title: A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

doi: 10.1038/s41598-021-89957-z

Figure Lengend Snippet: Tetrameric ACE2-Fc-TD out competes dimeric ACE2-Fc in viral neutralization assays. The neutralizing potency of tetrameric ACE2-Fc-TD Quad in inhibition of viral infection was examined in pseudovirus and live SARS-CoV-2 infection assays. ( A , B ) Lentiviral pseudotyped virus expressing SARS-CoV-2 spike protein were combined with SAD-S35 (AcroBiosystems), with ACE2-Fc, or with ACE2-Fc-TD and added to HEK 293 T/17 cells expressing ACE-2 and TMPRSS2 after 1 h. The cells were incubated for 60 h before luciferase activity, indicative of viral entry, was determined. ( C , D ) Further compares the neutralization of ACE2-Fc-TD, with H4-Fab-TD or with B38-Fab-TD. IC 50 data ( E ) were calculated using GraphPad Prism software and compare the efficacy of the two forms of soluble ACE2 proteins with the commercially available anti-SARS-CoV-2 antibody SAD-S35 and tetramerized anti-SARS-CoV-2 Fab antibodies H4 and B38 . ( F ) ACE2-Fc (dimeric for ACE2) and ACE2-Fc-TD (tetrameric for ACE2) proteins were preincubated with SARS-CoV-2 for 30 min at 37 °C. Vero E6 cells were infected with the ACE2-virus mixtures for 1 h at 37 °C before washing and continued incubation for 15 h at 37 °C. Cells were recovered and viral RNA was assayed by qRT-PCR. The graph shows relative fold change of viral RNA based on the Ct values compared to cellular RNA levels of mock infected cells. Data are represented as mean ± SD. (Student’s t test: ****P < 0.0001 p * < 0.05).

Article Snippet: 60 μg of pTT5-CoV-2 RBD plasmid DNA was added to 3 mL of Opti-Mem (Thermofisher). and 160ul of ExpiFectamine (Thermofisher) added to 3 mL of Opti-Mem and both incubated for 5 min at room temperature.

Techniques: Neutralization, Inhibition, Infection, Expressing, Incubation, Luciferase, Activity Assay, Software, Quantitative RT-PCR