tmprss2 activating protease  (ATCC)

Journal: Journal of Nanobiotechnology

Article Title: A bivalent spike-targeting nanobody with anti-sarbecovirus activity

doi: 10.1186/s12951-025-03243-y

Figure Lengend Snippet: Monovalent and bivalent 7F constructs neutralize authentic SARS-CoV-2 in A549 cells and HAE cell cultures and authentic SARS-CoV in Vero cells. A . Neutralization of SARS-CoV-2 in A549 ACE2+TMPRSS2+ cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting A549 ACE2+TMPRSS2+ cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV-2 infected cells. B . Neutralization of SARS-CoV-2 in HAE cell culture. HAE cultures were incubated with SARS-CoV-2 and 100 nM nanobodies or 10 µM remdesivir on the apical side for 2 h. Nanobody incubation was repeated every 24 h. Graph showing the quantification of viral replication in the cultures, evaluated by RT-qPCR. C . Neutralization of SARS-CoV in Vero cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting Vero cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV infected cells

Article Snippet: A549 cells ( Homo sapiens , lung carcinoma, ATCC CCL-185) expressing human ACE2 receptor protein and TMPRSS2 activating protease (A549 ACE2+TMPRSS2+ ) [ ], Vero cells ( Cercopithecus aethiops , kidney epithelial, ATCC CCL-81) and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS for A549 and Vero cells and 10% FBS for VeroE6 cells, 1 mM sodium pyruvate (Gibco), nonessential amino acids (Lonza), penicillin (100 IU/ml), and streptomycin (100 IU/ml).

Techniques: Construct, Neutralization, Virus, Incubation, Infection, Quantitative RT-PCR, Cell Culture

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: Donor differences in infection levels and immune response following SARS-CoV-2 infection of HBEC ALI cultures. HBEC ALI cultures from 8 different donors were infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side of cultures from (A) female donors, n = 4, and (B) male donors, n = 4, was quantified by qPCR at the indicated time points. (C) Heatmap displaying the mean expression (log 2 ) of ACE2 and TMPRSS2 in HBEC ALI cultures from each donor. (D) Volcano plot showing differentially expressed genes between uninfected (mock) and infected HBEC ALI cultures in group high. The statistical P value (–log 10 ) is plotted against the gene expression difference (log 2 ). Dotted lines highlight the significance cutoff at log fold changes of −1/1 (vertical line) and at a P value of 0.05 (horizontal line). (E) Heatmap displaying the significantly upregulated genes in group high upon infection. Shown are the mean expression difference compared to individual mock samples for each group (log 2 fold difference). (F and G) Cytokine levels in (F) apical and (G) basolateral samples collected at 72 h postinfection were analyzed using Proximity Extension Assay (Olink) and normalized to mock-treated samples for each donor individually. Mean values and standard error of the mean (SEM) are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01).

Article Snippet: Active recombinant TMPRSS2 was purchased from Cusabio (CSB-YP023924HU).

Techniques: Infection, Expressing, Gene Expression

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).

Article Snippet: Active recombinant TMPRSS2 was purchased from Cusabio (CSB-YP023924HU).

Techniques: Infection, Inhibition, Transfection, Expressing, Synthesized, SPR Assay, Binding Assay, Positive Control, Protease Inhibitor, Control, Recombinant, Enzyme-linked Immunosorbent Assay