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: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Overexpression of recombinant TMPRSS2 in HEK293T cells. ( A ) pcDNA3.1 empty vector, pcDNA3.1 eGFP , or pcDNA3.1 TMPRSS2+His plasmids (top panel) used to transfect HEK293T cells. Photomicrograph of pcDNA3.1 eGFP transfected cells (HEK293T eGFP ) (bottom panel). Cell lysates were prepared and immunoblotted with ( B ) anti-His antibody or ( C ) anti-TMPRSS2 antibody. ( D ) Immunocytochemistry staining to detect TMPRSS2 in control (HEK293T pcDNA3.1 ) and TMPRSS2–transfected HEK293T (HEK293T TMPRSS2 ) cells. ( E ) Spontaneous TMPRSS2 activity using the fluorogenic substrate Boc-QAR-AMC as a function of time in the HEK293T pcDNA3.1 and HEK293T TMPRSS2 cells. Data shown are representative or triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to HEK293T pcDNA3.1 cells.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Over Expression, Recombinant, Plasmid Preparation, Transfection, Immunocytochemistry, Staining, Control, Activity Assay

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of AAT. ( A ) HEK293T TMPRSS2 cells were left untreated or pre-treated with AAT at the indicated final concentrations or AEBSF (50 µM) for 30 min, followed by addition of the fluorogenic substrate Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. ( B ) TMPRSS2 activity at 90 min. Data shown are the triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min compared to untreated cells. AAT = alpha-1-antitrypsin.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of AAT alone or with UFH, nadroparin, or enoxaparin. HEK293T TMPRSS2 cells were pre-treated with AAT alone at the indicated concentrations or with ( A ) UFH, ( B ) nadroparin (Nadro), or ( C ) enoxaparin (Enox) at the indicated final concentrations for 30 min, followed by addition of the fluorescent substrate Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. ( D ) TMPRSS2 activity at 90 min in untreated cells and cells incubated with AAT ± two concentrations of nadroparin, UFH, and enoxaparin. Data shown are the triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min. Unfractionated heparin = UFH.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of UFH, nadroparin, or enoxaparin alone or with AAT. HEK293T TMPRSS2 cells were pre-treated with ( A ) UFH, ( B ) nadroparin, or ( C ) enoxaparin ± AAT at the indicated final concentrations for 30 min, followed by addition of the fluorescent substrate Boc-QAR-AMC. ( D ) HEK293T TMPRSS2 cells were grown in Gibco 293 Serum-free Medium II, pre-treated with enoxaparin at the indicated concentrations for 30 min, followed by addition of Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Modeling TMPRSS2 and its interaction with AAT and heparin. ( A ) Model of the TMPRSS2–AAT Michaelis complex. TMPRSS2 domain organization is shown in inset (TM = transmembrane region; residue numbers of domain boundaries are indicated). Domains are colored separately: low-density lipoprotein receptor A (LDLR-A, plum), scavenger receptor cysteine-rich repeat (SRCR, green), peptidase S1 (catalytic) domain (cyan). Disulphides are shown as sticks. A conserved disulphide formed between the catalytic domain and SRCR domain (244–365) is indicated. Scissors indicate the cleavage site at R255 at the N-terminus of the catalytic domain that generates the mature form. N- and C-termini are labeled. The catalytic triad (H296, D345 and S441) are shown as red sticks. TM region in lipid bilayer is also indicated as a schematic, indicating proximity to the plasma membrane, resembling the homologue hepsin. Reactive centre loop (RCL of AAT, bound to the active site of TMPRSS2 is labeled. ( B ) Molecular surface of TMPRSS2 showing bound RCL (magenta cartoon). Catalytic residues are labelled. P1 sidechain of AAT occupies the S1 site of TMPRSS2, superimposed on the TMPRSS2 inhibitor nafamostat (wheat). ( C ) Hydrogen-bonding interactions between the RCL of AAT and TMPRSS2. Black broken lines indicate hydrogen bonds. ( D ) Heparin molecules stabilize TMPRSS2–AAT association by acting as electrostatic bridges. Electrostatic potential surfaces of the TMPRSS2–AAT complex (blue = positive, red = negative) showing docked heparin 4mers binding to electropositive patches on the molecular surface. Carbon atoms of each 4mer heparin are coloured differently. ( E ) Location of Lys/Arg residues (sticks: AAT = magenta; TMPRSS2 = cyan) in the vicinity of heparin (grey sticks) binding sites at the TMPRSS2(cyan)–AAT(magenta) interface. Semi-transparent molecular surface is shown, and Lys/Arg residues that contribute to unfavorable electrostatics at the interface are shown and labelled (sidechain nitrogen atoms colored blue). ( F ) Poor electrostatic complementarity at P2′, P1′, P1, P2, P3, and P4 of the RCL (magenta) in the TMPRSS2 active site. P1 residue of the AAT RCL and cognate S1 pocket of TMPRSS2 are labeled. View is orthogonal to ( E ) showing the lysines located at the interface rim that are unfavorably close to positively charged residues of AAT.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Residue, Labeling, Clinical Proteomics, Membrane, Binding Assay

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Predicted binding free energies for representative protease-inhibitor complexes.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Binding Assay, Protease Inhibitor

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Proposed model for TMPRSS2–AAT–heparin ternary complexation at the cell surface, preventing activation of the S2 domain. Heparin (red) neutralizes the repulsive electrostatic forces at the TMPRSS2 (wheat)–AAT (blue) interaction rim, in accordance with the predicted binding mode shown in Figs. D,E and . AAT inhibition of elastase cleavage at the site created by the D614G mutation of the spike protein (B.1.1.7 variant of SARS-CoV-2) is also shown. The inset shows the possibility that one heparin molecule could bridge multiple TMPRSS2–AAT complexes.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activation Assay, Binding Assay, Inhibition, Mutagenesis, Variant Assay

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: (A) Scheme displaying the enzymatic assay principle for the fluorogenic peptide substrate. The fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC has low fluorescence compared to the fluorescent 7-amino-4-methylcoumarin (AMC), which is released upon proteolytic cleavage. The scissile bond is indicated in red. (B) Schematic of the truncated yeast-expressed recombinant TMPRSS2 used in the fluorogenic assay, containing the low-density lipoprotein receptor A (LDLRA) domain, scavenger receptor cysteine-rich (SRCR) domain, and protease domain.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Enzymatic Assay, Fluorescence, Recombinant

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Assay performance for primary screening of the compound libraries in the TMPRSS2 fluorogenic biochemical activity assay. Z′ scores and signal-to-background values are plotted as data points for each plate. There were 32 positive and 64 negative control wells on each 1536-well plate. (A) NCATS Pharmaceutical Collection (NPC), 2,678 compounds, (B) Mechanism Interrogation Plate (MIPE) library, 2,480 compounds, and (C) protease inhibitor library (PIL), 872 compounds. Black horizontal lines represent the mean values.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Activity Assay, Negative Control, Protease Inhibitor

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Label-free mass spectrometry biochemical assay. (A) Peptide derived from the known S2′ cleavage site of SARS-CoV-2 spike (S) protein. (B) Scheme displaying the enzymatic assay principle for the unlabeled peptide substrate. The unlabeled peptide substrate, Cbz-SKPSKRFIED, is cleaved by TMPRSS2 to create two cleavage products, Cbz-SKPSKR and SFIED. The scissile bond is indicated in red. (C) Initial velocity ( V 0 ) was calculated for each substrate concentration by plotting product formation versus time. (D) The V 0 for each concentration were plotted against the various substrate concentrations to obtain the V max and K m . V max : 1.95 μmol/min and K m : estimated 2320 μM. (E) Comparison of TMPRSS2 fluorogenic detection and mass spectrometry detection assays by assessing dose–response inhibition by camostat and gabexate. (F) Mass spectrometry traces of the cleavage product ( m / z : 418.8/702.4) showing that addition of camostat prevents product formation in dose–response. Each peak is labeled with the concentration of camostat (nM) for that condition. (G) Assay performance from the mass spectrometry detection assay when screening the hits identified from the primary screening. Z′ of 0.71 and S/B of 7.37. (H) Dose–response inhibition of 7-hydroxycoumarin against TMPRSS2 in both fluorogenic and mass spectrometry detection assays showing the fluorescent molecule as a false-positive hit in the fluorogenic assay, but not interfering with the mass spectrometry detection assay.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Mass Spectrometry, Derivative Assay, Enzymatic Assay, Concentration Assay, Comparison, Inhibition, Labeling, Detection Assay

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Detailed TMPRSS2 Fluorogenic Biochemical Assay Protocol for qHTS

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Control, Incubation, Fluorescence

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Detailed TMPRSS2 Mass Spectrometry Detection Biochemical Assay

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Mass Spectrometry, Control, Incubation

Journal: Frontiers in Immunology

Article Title: Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

doi: 10.3389/fimmu.2021.714027

Figure Lengend Snippet: Effect of P. aeruginosa infection on ACE2 , TMPRSS2 , and FURIN expression in primary hAECs. Heatmap of ACE2 , TMPRSS2 , and FURIN expression (fold-change) (A) and kinetics of TMPRSS2 expression (shown in reads) (B) in primary hAECs isolated from non-CF and CF patients and infected with P. aeruginosa (multiplicity of infection = 0.25) [RNA-seq data extracted from a previous study , Benjamini-Hochberg adjusted p value (*p)]. (C) ACE2 and TMPRSS2 mRNA expression (in arbitrary units, a.u.) in submerged non-CF (n=4) and CF (n=4) primary hAECs stimulated with control medium (reference group) or Pa -F (50 ng/mL) for 6 h (ANOVA with Bonferroni’s multiple-comparison test, * P < 0.05).

Article Snippet: Real-time qPCR was performed by using an ABI QS3 with a Sensifast Probe Lo-Rox Kit (Bio-technofix, Guibeville, France), TaqMan probes for ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), FURIN (Hs00965485_g1), and GAPDH (Hs02786624_g1), and a cDNA template.

Techniques: Infection, Expressing, Isolation, RNA Sequencing Assay

Journal: Frontiers in Immunology

Article Title: Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

doi: 10.3389/fimmu.2021.714027

Figure Lengend Snippet: Effect of TLR5 and p38 inhibition on flagellin induced-TMPRSS2 expression in CFTR -deficient cells. (A) TMPRSS2 mRNA expression in Calu-3- CFTR -KD grown at the air-liquid interface and either not stimulated or stimulated for 6 h with 50 ng/mL ultrapure flagellin from S. Typhimurium ( St )-F, recombinant St -F, vaccigrade St - F , or Pa -F ( n = 3, ANOVA with Dunnett’s multiple-comparison test, * P < 0.05). (B) TMPRSS2 mRNA expression in Calu-3- CFTR -KD cells grown at the air-liquid interface and incubated for 1 h with isotype control (reference) or anti-TLR5 antibody (10 μg/mL) and then either not stimulated or stimulated for 6 h with Pa -F (50 ng/mL) ( n = 3, ANOVA with Dunnett’s multiple-comparison test, control group: Isotype/ Pa -F, * P < 0.05). TMPRSS2 mRNA expression in Calu-3- CFTR -KD cells grown at the air-liquid interface (C) or in primary CF hAECs (D) that were preincubated for 1 h with 20 μmol/L p38 or 20 μmol/L NF-κB inhibitors and then stimulated for 6 h with 50 ng/mL Pa -F in the presence of the inhibitor. ANOVA with Dunnett’s multiple-comparison test, control group: -/ Pa -F, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (E) Western blot (with 10 μg of protein) of phospho- and total p38 and phospho- and total NF-κB in primary hAECs stimulated with 50 ng/mL Pa -F.

Article Snippet: Real-time qPCR was performed by using an ABI QS3 with a Sensifast Probe Lo-Rox Kit (Bio-technofix, Guibeville, France), TaqMan probes for ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), FURIN (Hs00965485_g1), and GAPDH (Hs02786624_g1), and a cDNA template.

Techniques: Inhibition, Expressing, Recombinant, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

doi: 10.3389/fimmu.2021.714027

Figure Lengend Snippet: Effect of P. aeruginosa flagellin on ACE2, FURIN, and TMPRSS2 expression in CFTR -deficient Calu-3 cells. (A) ACE2 and TMPRSS2 mRNA expression in submerged cultures of Calu-3, Beas-2B (reference), and 16HBE14o- cell lines ( n = 3, ANOVA with Dunnett’s multiple-comparison test, control group: Calu-3, **** P < 0.0001). GAPDH , housekeeping gene. Representative western blot (with 20 μg of protein) showing ACE2 and β-actin protein expression in submerged cultures of Calu-3, Beas-2B, and 16HBE14o- cell lines. TMPRSS2 (B) and ACE2 (C) mRNA expression (relative to that of housekeeping gene GAPDH ) in Calu-3- CFTR -WT (reference group) and - CFTR -KD cells grown at the air-liquid interface and either not stimulated (–) or stimulated for 3 or 6 h with P. aeruginosa flagellin ( Pa -F, 50 ng/mL) ( n = 5, ANOVA with Bonferroni’s multiple-comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). (D) Immunofluorescence analysis of TMPRSS2 and ACE2 protein expression in Calu-3 cells (ATCC) grown at the air-liquid interface and stimulated with Pa -F for 18 h; scale bar, 20 μm.

Article Snippet: Real-time qPCR was performed by using an ABI QS3 with a Sensifast Probe Lo-Rox Kit (Bio-technofix, Guibeville, France), TaqMan probes for ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), FURIN (Hs00965485_g1), and GAPDH (Hs02786624_g1), and a cDNA template.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

doi: 10.3389/fimmu.2021.714027

Figure Lengend Snippet: Effect of TMPRSS2 induction by P. aeruginosa flagellin on SARS-CoV-2 infectivity in CFTR -deficient Calu-3 cells. (A) Intracellular nsp14/GAPDH and (B) apical (supernatant) nsp14 mRNA expression in Calu-3- CFTR -WT (reference group) and - CFTR -KD cells grown at the air-liquid interface and either not stimulated (black circle) or stimulated (red circle) for 16 h with Pa -F 50 ng/mL), and then infected for 24 h with SARS-CoV-2 (multiplicity of infection = 1) ( n = 3, ANOVA with Bonferroni’s multiple-comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Real-time qPCR was performed by using an ABI QS3 with a Sensifast Probe Lo-Rox Kit (Bio-technofix, Guibeville, France), TaqMan probes for ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), FURIN (Hs00965485_g1), and GAPDH (Hs02786624_g1), and a cDNA template.

Techniques: Infection, Expressing

Journal: Frontiers in Immunology

Article Title: Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

doi: 10.3389/fimmu.2021.714027

Figure Lengend Snippet: Schematic illustrating the results. P. aeruginosa interacts with the airway epithelial cells of CF patients, notably by activating the TLR5 signaling pathway through its virulence factor, flagellin. This activation, dependent on p38 MAPK and NF-kB, leads to an increase in TMPRSS2 which could regulate SARS-CoV2 infectivity.

Article Snippet: Real-time qPCR was performed by using an ABI QS3 with a Sensifast Probe Lo-Rox Kit (Bio-technofix, Guibeville, France), TaqMan probes for ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), FURIN (Hs00965485_g1), and GAPDH (Hs02786624_g1), and a cDNA template.

Techniques: Activation Assay, Infection

Journal: Journal of Medicinal Chemistry

Article Title: Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L

doi: 10.1021/acs.jmedchem.1c01494

Figure Lengend Snippet: Targeting cathepsin L with gallinamide A to inhibit SARS-CoV-2. (A) Role of host proteases in cell entry mechanisms for SARS-CoV-2 (endocytic entry route shown, NB: TMPRSS2 primes S-protein for nonendocytic entry). (B) Structure of the natural product depsipeptide CatL inhibitor gallinamide A ( 1 ). (C) Enzymatic activity of CatL, Mpro, and PLpro following incubation with 10 μM gallinamide A. Assays were performed in triplicate wells, data are the means +/- SD, and relative activity was compared to reactions containing 0.2% DMSO.

Article Snippet: Recombinant human TMPRSS2 was purchased from Cusabio Technology (CSB-YP023924HU) and assayed with 86 μM Boc-QAR-AMC in 25 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl 2 , 0.01% Triton X-100, pH 8.0 at a final concentration of 3 nM.

Techniques: Activity Assay, Incubation

Journal: Journal of Medicinal Chemistry

Article Title: Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L

doi: 10.1021/acs.jmedchem.1c01494

Figure Lengend Snippet: Gallinamide A ( 1 ) inhibits viral entry into VeroE6 cells and synergizes with TMPRSS2-inhibition to increase anti-SARS-CoV-2 efficacy. (A) Time of addition study to establish the kinetics of gallinamide A inhibitory activity on SARS-CoV-2 infection. Gallinamide A (0.5 μM) was added at the times indicated prior to or after infection (spinoculation) of VeroE6 cells. At 24 h postinfection, cells were stained with antinucleocapsid antibody (green) and Hoechst dye (blue) to determine the percentage of infected cells. Representative images for the various test conditions are shown, scale bar 100 μm. (B) Inhibition of SARS-CoV-2 infection by gallinamide A ( 1 ), in comparison to and in synergism with the TMPRSS2 protease inhibitor nafamostat mesylate, directly compared in ACE2/TMPRSS2 expressing HEK293 cells. Data are the means ± SD and are representative of three independent replicates.

Article Snippet: Recombinant human TMPRSS2 was purchased from Cusabio Technology (CSB-YP023924HU) and assayed with 86 μM Boc-QAR-AMC in 25 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl 2 , 0.01% Triton X-100, pH 8.0 at a final concentration of 3 nM.

Techniques: Inhibition, Activity Assay, Infection, Staining, Comparison, Protease Inhibitor, Expressing

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: A Scatterplots indicating enriched genes related with serine-type peptidase. B PPI network of DEGs linked with serine-type peptidase indicates the protein – protein interaction with Serpine1 and Serpine2 (PP protein – protein). C Heatmap depicting the expression levels of genes related with serine-type peptidase inhibitor activity differentially expressed in S. epidermidis– inoculated NHNE cells depending on a cellular subset. D Serpine1 mRNA and E protein levels were monitored by real-time PCR and western blot analysis following S. epidermidis inoculation at 1 dpi with transfection with cont shRNA and uPA shRNA. F Serpine1 (blue bar) and PLAU mRNAs (red bar) were compared in NHNE cells after S. epidermidis inoculation. G ELISA results showed the secreted protein levels of Serpine1 (blue bar) and uPA (red bar) in S. epidermidis -inoculated NHNE cells. H The intracellular protein levels of HAT and Serpine1 were measured in the cell lysate of S. epidermidis– inoculated NHNE cells at 4, 8, and 24 h using western blot analysis. I The mRNA levels of proteases, such as TMPRSS2, TMPRSS11E, TMPRSS11F, and KLK5, which were targeted by Serpine1, were measured using the cell lysate of S. epidermidis -inoculated NHNE cells at 4, 8, and 24 h. Western blot results are representative of five independent experiments. Real-time PCR and plaque assay results are presented as mean ± SD values from five independent experiments. * p < 0.05 vs. mock-infected NHNE cells.

Article Snippet: Primers for human or mouse Serpine1 (assay ID Hs00167155_m1, Mm00435858_m1), Serpine2 (assay ID Hs00299953_m1, Mm00436753_m1), TMPRSS11E (assay ID Hs01070171_m1, Mm01212186_m1), TMPRSS11F (assay ID Hs01592083_m1, Mm00812591_m1), TMPRSS2 (assay ID Hs01122322_m1, Mm00443687_m1), and KLK5 (assay ID Hs01548153_m1, Mm01203811_m1) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Plaque Assay, Infection

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: Differential expression gene (DEG) linked with serine-type peptidase in S. epidermidis -inoculated NHNE cells.

Article Snippet: Primers for human or mouse Serpine1 (assay ID Hs00167155_m1, Mm00435858_m1), Serpine2 (assay ID Hs00299953_m1, Mm00436753_m1), TMPRSS11E (assay ID Hs01070171_m1, Mm01212186_m1), TMPRSS11F (assay ID Hs01592083_m1, Mm00812591_m1), TMPRSS2 (assay ID Hs01122322_m1, Mm00443687_m1), and KLK5 (assay ID Hs01548153_m1, Mm01203811_m1) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Quantitative Proteomics

Journal: Cell

Article Title: Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike

doi: 10.1016/j.cell.2022.04.035

Figure Lengend Snippet: Virological features of BA.2 in vitro (A) Scheme for the chimeric recombinant SARS-CoV-2 used in this study. The SARS-CoV-2 genome and its genes are shown. The template was SARS-CoV-2 strain WK-521 (PANGO lineage A, GISAID ID: EPI_ISL_408667), and the S genes were swapped with those of the respective lineages/strains (GISAID IDs are indicated in the figure). ORF7a was swapped with the sfGFP gene. (B) Growth kinetics of chimeric recombinant SARS-CoV-2 in Vero cells, VeroE6/TMPRSS2 cells, Calu-3 cells, and human nasal epithelial cells. (C) Fluorescence microscopy. The GFP area was measured in infected VeroE6/TMPRSS2 cells (multiplicity of infection [m.o.i.] 0.01) at 48 h.p.i. Left, representative panels. Higher-magnification views of the regions indicated by squares are shown at the bottom. Representative time-course data are shown in Figure S2 E. Scale bars, 500 μm. Right, the summarized results. The numbers in the panel indicate the numbers of GFP-positive cells counted. (D) Plaque assay. Representative panels (left) and a summary of the recorded plaque diameters (20 plaques per virus) (right) are shown. (E) S expression on the cell surface. Representative histograms stained with an anti-S1/S2 polyclonal antibody (left) and the summarized data (right) are shown. In the left panel, the number in the histogram indicates mean fluorescence intensity (MFI). Gray histograms indicate isotype controls. (F) S-based fusion assay. The recorded fusion activity (arbitrary units) is shown. (G) Western blotting. Left, representative blots of S-expressing cells (top) and pseudovirus (bottom). ACTB is an internal control for the cells, whereas HIV-1 p24 is an internal control for the pseudovirus. kDa, kilodalton. Middle, the ratio of S2 to the full-length S plus S2 proteins in the cells. Right, the ratio of S2 to HIV-1 p24 in the pseudovirus (supernatant). (H) Pseudovirus assay. The percent infectivity compared with that of the virus pseudotyped with B.1.1 S are shown. (I) Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. The percentage of SARS-CoV-2 S RBD expressed on yeast binding to soluble ACE2 (left) and the summarized K D values (right) are shown. (J) TMPRSS2 expression on the cell surface. Representative histograms stained with an anti-TMPRSS2 polyclonal antibody (left) and the summarized data (right) are shown. In the left panel, the number in the histogram indicates MFI. Gray histograms indicate the isotype controls. (K) S-based fusion assay. The recorded fusion activity (arbitrary units) is shown. (L) Fold increase in pseudovirus infectivity based on TMPRSS2 expression. (M) E64d treatment. IC50, 50% inhibitory concentration; ND, not determined. (N) Growth kinetics of chimeric recombinant SARS-CoV-2 in HK293-ACE2 and HEK293-ACE2/TMPRSS2 cells. Assays were performed in quadruplicate (B, H, L, J, and N), octuplicate (B, most right) or triplicate (E–G, I, J, K, and M), and the presented data are expressed as the average ± SD. Each dot indicates the result of an individual plaque (D) and an individual replicate (E, G– J, L and I). Statistically significant differences between BA.2 and other variants across time points were determined by multiple regression (B, F, K, and N). Family-wise error rates (FWERs) calculated using the Holm method are indicated in the figures. Statistically significant differences between BA.1 and BA.2 were determined by two-sided Mann-Whitney U tests (C and D), two-sided Student’s t tests (E, H, and I), or two-sided paired t test (G). See also Figure S3 .

Article Snippet: Rabbit anti-TMPRSS2 polyclonal antibody , BIOSS , Cat# BS-6285R; RRID: AB_11102333.

Techniques: In Vitro, Recombinant, Fluorescence, Microscopy, Infection, Plaque Assay, Expressing, Staining, Single Vesicle Fusion Assay, Activity Assay, Western Blot, Binding Assay, Concentration Assay, MANN-WHITNEY

Journal: Cell

Article Title: Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike

doi: 10.1016/j.cell.2022.04.035

Figure Lengend Snippet: Virological features of BA.2 in vitro , related to Figure 3 (A) Fluorescence microscopy. The GFP area were measured in infected VeroE6/TMPRSS2 cells (m.o.i. 0.01) at 24, 48, and 72 h.p.i. Higher-magnification views of the regions indicated by squares are shown at the bottom. The panels at 48 h.p.i. are identical to those shown in Figure 3 C. (B) Coculture of S-expressing cells with HEK293-ACE2/TMPRSS2 cells. Left, representative images of S-expressing cells cocultured with HEK293 cells (top) or HEK293-ACE2/TMPRSS2 cells (bottom). Nuclei were stained with Hoechst 33342 (blue). Right, size distribution of syncytia (green). The numbers in the panel indicate the numbers of GFP-positive syncytia counted. (C) Cytotoxicity of E64d in HOS-ACE2-TMPRSS2 cells. The cells were cultured in the presence of serially diluted E64d for 48 h, and the cytotoxicity was measured using a cell counting kit-8 solution. The assay for each concentration was performed in sextuplicate, and the data are expressed as the average ± SD. CC50, 50% cytotoxic concentration. Scale bars, 500 μm (A) or 200 μm (B).

Article Snippet: Rabbit anti-TMPRSS2 polyclonal antibody , BIOSS , Cat# BS-6285R; RRID: AB_11102333.

Techniques: In Vitro, Fluorescence, Microscopy, Infection, Expressing, Staining, Cell Culture, Cell Counting, Concentration Assay

Journal: Cell

Article Title: Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike

doi: 10.1016/j.cell.2022.04.035

Figure Lengend Snippet:

Article Snippet: Rabbit anti-TMPRSS2 polyclonal antibody , BIOSS , Cat# BS-6285R; RRID: AB_11102333.

Techniques: Recombinant, Expressing, Protease Inhibitor, Activity Assay, Luciferase, CCK-8 Assay, Sequencing, Plasmid Preparation, Software

Journal: Translational Cancer Research

Article Title: Analysis of novel enzalutamide-resistant cells: upregulation of testis-specific Y-encoded protein gene promotes the expression of androgen receptor splicing variant 7

doi: 10.21037/tcr-20-1463

Figure Lengend Snippet: Characterization of AR status and expression of AR target genes in SAS MDV No. 3–14 cells. (A) LNCaP and SAS MDV No. 3–14 cells were cultured under androgen-deprived conditions for 1 day. Total RNA was extracted from cells and cDNA was synthesized. The mRNA expression of AR , AR-v7 , and GAPDH was analyzed by qRT-PCR analysis. The expression of AR and AR-v7 was normalized to the corresponding expression of GAPDH , and the relative mRNA expression in LNCaP cells was set as 1. Data represent mean ± SD (N=3). (B) Whole-cell lysates were harvested and subjected to immunoblotting analysis. An AR antibody recognizing the AR n-terminal domain (AR D6F11), an AR-v7 specific antibody, and a GAPDH antibody were used for protein detection. (C) The mRNA expression of KLK3 , FKBP5 , TMPRSS2 , CDK1 , CDC20 , and UBE2C in LNCaP or SAS MDV No. 3–14 cells was analyzed by qRT-PCR analysis. The level of each target gene was normalized to the corresponding expression of GAPDH, and relative mRNA expression in LNCaP cells was set as 1. Data represent mean ± SD (N=3). ***, P<0.001. AR, androgen receptor; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; KLK3 , kallikrein-3; FKBP5 , FKBP prolyl isomerase 5; TMPRSS2 , transmembrane serine protease 2; CDK1 , cyclin-dependent kinase 1; CDC20 , cell division cycle 20; UBE2C , ubiquitin conjugating enzyme E2 C .

Article Snippet: Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using the Applied Biosystems 7900HT Fast Real-Time PCR System and TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) for AR (Hs00171172_m1), AR - v7 (Hs04260217_m1), glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ; Hs99999905_m1), kallikrein-3 ( KLK3 ; Hs02576345_m1), FKBP prolyl isomerase 5 ( FKBP5 ; Hs01561006_m1), transmembrane serine protease 2 ( TMPRSS2 ; Hs01120965_m1), ubiquitin-conjugating enzyme E2 C ( UBE2C ; Hs00964100_g1), cyclin-dependent kinase 1 ( CDK1 ; Hs00938777_m1), cell division cycle 20 ( CDC20 ; Hs00426680_mH), and TSPY (Hs00413986_m1).

Techniques: Expressing, Cell Culture, Synthesized, Quantitative RT-PCR, Western Blot, Ubiquitin Proteomics

Journal: Translational Cancer Research

Article Title: Analysis of novel enzalutamide-resistant cells: upregulation of testis-specific Y-encoded protein gene promotes the expression of androgen receptor splicing variant 7

doi: 10.21037/tcr-20-1463

Figure Lengend Snippet: The function of TSPY in the proliferation, expression and transcriptional activity of AR in SAS MDV No. 3–14 cells. (A) The mRNA expression of TSPY in indicated cell lines was analyzed by qRT-PCR analysis. The expression of the TSPY gene was normalized to the corresponding expression of GAPDH, and relative mRNA expression in LNCaP cells was set as 1. Data represent mean ± SD (N=3). (B) Whole- cell lysates of indicated cell lines were harvested and subjected to immunoblotting analysis. The TSPY and GAPDH antibodies were used as primary antibodies, respectively. (C) SAS MDV No. 3–14 cells were transfected with negative control (NGT) or TSPY siRNA under androgen-deprived conditions. Three days after transfection with siRNA, cell proliferation was evaluated using the CellTiter-Glo assay. Data represent mean ± SD (N=3). (D,E) Three days after transfection with siRNA, whole-cell lysates were harvested and subjected to immunoblotting analysis. An AR antibody recognizing the AR n-terminal domain (AR D6F11), an AR-v7 specific antibody, a TSPY antibody, and a GAPDH antibody were used as primary antibodies. The expression of TSPY , AR , AR-v7 , KLK3 , FKBP5 , TMPRSS2 , CDK1 , CDC20 , and UBE2C genes was analyzed by qRT-PCR analysis. The expression of each gene was normalized to the corresponding expression of GAPDH, and relative mRNA expression in the NGT-treatment group was set as 1. Data represent mean ± SD (N=3). *, P<0.05; **, P<0.01; ***, P<0.001. n.s., not significant; AR, androgen receptor; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; NGT, negative control; KLK3 , kallikrein-3; FKBP5 , FKBP prolyl isomerase 5; TMPRSS2 , transmembrane serine protease 2; CDK1 , cyclin-dependent kinase 1; CDC20 , cell division cycle 20; TSPY , testis-specific Y-encoded protein; UBE2C , ubiquitin conjugating enzyme E2 C .

Article Snippet: Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using the Applied Biosystems 7900HT Fast Real-Time PCR System and TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) for AR (Hs00171172_m1), AR - v7 (Hs04260217_m1), glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ; Hs99999905_m1), kallikrein-3 ( KLK3 ; Hs02576345_m1), FKBP prolyl isomerase 5 ( FKBP5 ; Hs01561006_m1), transmembrane serine protease 2 ( TMPRSS2 ; Hs01120965_m1), ubiquitin-conjugating enzyme E2 C ( UBE2C ; Hs00964100_g1), cyclin-dependent kinase 1 ( CDK1 ; Hs00938777_m1), cell division cycle 20 ( CDC20 ; Hs00426680_mH), and TSPY (Hs00413986_m1).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Glo Assay, Ubiquitin Proteomics