Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Immunohistochemistry confirms positive staining for ACE2 in several human tissue types. Representative fluorescent images of 4% formaldehyde fixed human tissue sections (n ≥ 3 independent donors, stained in duplicate) treated with ACE2 poly antibody (R&D AF933), visualised in green, and Hoechst 33342 nuclear stain, visualised in blue. Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney, with cortex and medulla indicated; ( b ) Kidney control section treated with secondary antibody and Hoechst 33342 alone; ( c ) Heart tissue, comprised predominantly of cardiomyocytes; ( d ) Lung, with preserved airway structures; ( e ) Liver, comprised predominantly of hepatocytes with preserved bile duct structures; ( f ) Hepatic artery section.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Schematic showing the critical protein domains of full-length ACE2 versus the short dACE2 isoform. The 805 amino acid full-length ACE2 protein (left) is comprised of an extracellular domain that protrudes into the extracellular (E.C.) space and an intracellular domain that remains in the intracellular (I.C.) space. The extracellular domain is made up of a signal peptide (SP) that extends from positions 1–18; the peptide-binding catalytic site that covers 272–515; two spike protein binding sites (SB) located at 24–42 and 353–357; a collectrin-like domain (CLD) that covers 616–805; and a short transmembrane domain (TMD) that spans the membrane at positions 741–762. The short dACE2 isoform (right) loses all amino acids up to positon 357 and a unique 10 amino acid sequence caps the N-terminus. Note that dACE2 has lost both its spike protein binding sites and the catalytic site is non-functional. The diagram also shows the potential binding sites for the ACE2 poly antibody (green), raised against an 18–740 amino acid immunogen of ACE2, versus the single proprietary binding site that sits between amino acids 200–300 for the ACE2 mono antibody (orange). If the full ACE2 isoform is present, green and orange fluorescent signal will be observed in immunological staining studies. If only the short ACE2 isoform is present, green fluorescent signal alone will be observed due to the lack of the monoclonal antibody binding site. The schematic was generated using templates from Servier Medical Art (smart.servier.com).

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Binding Assay, Protein Binding, Sequencing, Functional Assay, Staining, Generated

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Differential expression of full-length ACE2 and the short dACE2 isoform in a panel of human tissues. Representative fluorescent images (n = 3 independent donors, stained in duplicate) of human tissue fixed in 4% formaldehyde and treated with ACE2 poly (left column, visualised in green) and ACE2 mono (centre column, visualised in orange). Merged images (right column, overlay) also include Hoechst 33342 nuclear marker (visualised in blue). Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney cortex, with a glomerulus (glom.) indicated; ( b ) Kidney border, showing a region where tubules of the cortex meet tubules of the medulla; ( c ) Heart tissue, showing cardiomyocytes; ( d ) Lung tissue, showing an airway structure; ( e ) Liver bile duct; ( f ) Liver hepatocytes.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Expressing, Staining, Marker

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Quantification of differential expression of full-length ACE2 and the short dACE2 isoform in specific structures of human tissues. Graphical output shows the fold change in mean fluorescence observed for the respective secondary antibodies used to visualise ACE2 poly and ACE2 mono in distinct anatomical structures of the human tissue sections shown in Fig. . For each structure, n = 6 from ≥ 2 tissue sections. ( a ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in kidney cortex and glomeruli (Glom.), versus the normalised mean fluorescence observed in the renal medulla. ( b ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in airway structures and blood vessels (Blood ves.) of the lung, versus the normalised mean fluorescence observed in the connective tissue (Connec.). ( c ) Fold change in mean fluorescence observed for ACE2 poly and ACE2 mono in liver bile ducts and blood vessels (Blood ves.), versus the normalised mean fluorescence observed in liver hepatocytes (Hepato.). All data show mean ± SEM with individual data points shown. Statistical analyses of data included a one way ANOVA with multiple comparisons using Tukey’s correction. Statistical significance was determined where p < 0.05. **** or #### = p < 0.0001; ** = p < 0.01; n.s. = no significant difference.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Differential expression in humans of the viral entry receptor ACE2 compared with the short delta ACE2 isoform lacking SARS-CoV-2 binding sites

doi: 10.1038/s41598-021-03731-9

Figure Lengend Snippet: Binding of fluorescent SARS-CoV-2 spike-AF647 in ACE2 positive cells in a panel of human tissues. Representative confocal fluorescent images (n ≥ 2 independent donors, stained in duplicate) of human tissue fixed in 4% formaldehyde and treated with ACE2 poly (left column, visualised in green) and 1 µM spike-AF647 (centre column, visualised in red). Merged images (right column, overlay) also include Hoechst 33342 nuclear marker (visualised in blue). Scale bars are as indicated in figure. Tissues shown include: ( a ) Kidney cortex; ( b ) Kidney medulla; ( c ) Left ventricle heart tissue, showing cardiomyocytes; ( d ) Lung tissue, showing an airway structure; ( e ) Liver bile duct; ( f ) Liver hepatocytes.

Article Snippet: Sections were then incubated overnight at 4 °C with ACE2 poly primary goat polyclonal antibody to Human ACE2 (AF933; R&D; 1:100) and/or ACE2 mono primary rabbit monoclonal antibody to a site between 200 and 300 amino acids (N-terminus) of Human ACE2 (ab108252; Abcam; 1:100), depending on the experiment.

Techniques: Binding Assay, Staining, Marker

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Reporter gene fusion assay. Donor cells (293T) were co-transfected with plasmids encoding the T7 polymerase together with either the empty vector (negative control) or a viral envelope protein i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus glycoprotein (VSV-G). The viral envelope proteins contain authentic signal peptides and transmembrane domains to target to the plasma membrane. Although the SARS-CoV-2 spike protein was localized to the endoplasmic reticulum-Golgi intermediate compartment, leaky transport would still target a proportion of the spike protein to the plasma membrane. Target cells (either 293T-ACE2 or 293T-ACE2-TMPRSS2) were co-transfected with two reporter genes expressing the luciferase protein under the T7 promoter and the internal control, β-galactosidase under the CMV promoter. Binding of the spike protein with the angiotensin-converting enzyme 2 (ACE2) protein will trigger fusion of the donor and target cells. Mixing of cytoplasmic contents enabled the T7 polymerase from the donor cells to bind to the T7 promoter in the target cells to drive transcription of the luciferase gene. The transcript was then translated from the encephalomyocarditis (EMCV) internal ribosome entry site (IRES) element to produce a luciferase read-out. Fusion was measured as a ratio of the luciferase activity to the β-galactosidase activity.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Single Vesicle Fusion Assay, Transfection, Plasmid Preparation, Negative Control, Expressing, Luciferase, Binding Assay, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Vesicular stomatitis virus glycoprotein triggers universal acidic pH fusion. (A,B) Donor 293T cells were transfected with empty vector (negative control) or the vesicular stomatitis glycoprotein (VSV-G) together with a plasmid encoding the T7 polymerase. 19.000 donor cells were co-cultured with 19,000 target cells (A) (293T, 293T-ACE2, 293T-ACE2-TMPRSS2) or (B) Vero cells transfected with the luciferase and β-galactosidase reporter genes. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to their respective vector control in the respective cell type and fusion condition. Data are presented as mean +/− SD of 3 repeats. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Donor 293T cells transfected with 0.15 μg of VSV-G (left and right) or 0.074 μg of VSV-G (middle) were co-cultured with target 293T cells and fusion triggered by incubating with pH7.4 (left) or pH5 (middle and right) fusion buffers. Cells were fixed and stained with methylene blue. A syncytium was outlined. Fusion index was calculated as (S–N)/T where S = number of nuclei in syncytia; N = number of syncytia; T = total number of nuclei. All photomicrographs are of the same magnification and scale.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Plasmid Preparation, Negative Control, Cell Culture, Luciferase, Activity Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Severe acute respiratory syndrome coronavirus 2 spike protein triggers fusion in different cell types and fusion conditions. (A,B) Donor 293T cells were transfected with empty vector (negative control) or the severe acute respiratory syndrome coronavirus 1 and 2 spike proteins (SARS-1-S, SARS-2-S), respectively together with a plasmid encoding the T7 polymerase. 19,000 donor cells were co-cultured with 19,000 target cells (A) (293T, 293T-ACE2, 293T-ACE2-TMPRSS2) or (B) Vero cells transfected with the luciferase and β-galactosidase reporter genes. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to their respective vector control in the respective cell type and fusion condition. Data on the vesicular stomatitis virus glycoprotein (VSV-G) from are included for comparison purpose. Data are presented as mean +/− SD of 3 repeats. (C) Donor 293T cells transfected with an empty vector or serial doses of the plasmid expressing the SARS-2-S, as indicated, were co-cultured with target 293T-ACE2 cells under various fusion condition ( n = 1). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Plasmid Preparation, Negative Control, Cell Culture, Luciferase, Activity Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Syncytia formation is enhanced by TMPRSS2 or trypsin. 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were co-cultured with 19,000 donor 293T cells transfected with an empty vector or the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-2-S) together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Photomicrographs illustrate intact single cells and syncytia (arrowheads). Bright-field images are of the same magnification x100 and scale and from the same repeat.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase, Cell Culture, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Specificity of different modes of fusion. (A) 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were pre-incubated with 10 μg/ml of anti-spike monoclonal antibody (ACROB SPD-M128), 75 μg/ml soybean trypsin inhibitor (SBTi), 10 μg/ml leupeptin or 10 μM of individual drugs before co-cultured with 19,000 donor 293T cells transfected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins (SARS-2-S) together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to the solvent control in the respective cell type and fusion condition which is set as 1. Data are presented as mean+/-SD of >3 repeats. (B) Photomicrographs of fusion cell morphology. 50,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) pre-incubated with 28 μg/ml anti-spike monoclonal antibody (Sino Biol, 40592-R001) or 10 μM of individual drugs were co-cultured with 50,000 donor 293T cells transfected with an empty vector or the SARS-CoV-2 spike protein. The co-cultures were treated as indicated. Cells were fixed and stained with methylene blue. Bright-field images are of the same magnification x100 and scale. Fields of entire or mostly syncytia formation are framed with red squares. Fields of entire or mostly intact cells are framed with orange squares. Scattered syncytia are circled or outlined red and labelled S. Scattered intact cells are circled or outlined orange and labelled C. Sporadic intact cells and syncytia are marked by orange and red arrowheads, respectively. (C) Viability was measured by XTT assays in fusion cells (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) under physiological pH and treated with compounds as in (A) . Data are expressed as % viability to solvent control which is set as 100%. Data are presented as mean+/-SD of three repeats. (D) Negative screen. One of the three cell types: 293T, 293T-ACE2 and 293T-ACE2-TMPRSS2 (30,000 cells) was co-transfected with plasmids encoding the T7 polymerase, luciferase and β-galactosidase reporter genes and then treated with compounds as in (A) . Luciferase, β-galactosidase and the luciferase/β-galactosidase ratio are presented as ratios to the solvent controls which are set as 1. Data are presented as mean+/-SD of three repeats * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase, Incubation, Cell Culture, Plasmid Preparation, Activity Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Heatmap of drug inhibition profiles of different modes of fusion. 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were pre-incubated with 10 μM of individual drugs before co-cultured with 19,000 donor 293T cells transfected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to the solvent control in the respective cell type and fusion condition which is set as 1. Data are presented as mean of 3 repeats. Viability was measured by XTT assays in fusion cells (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) under physiological pH and expressed as % viability to solvent control which is set as 100%. Data are presented as mean of three repeats. Data outside the range are depicted as dark purple.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Inhibition, Transfection, Luciferase, Incubation, Cell Culture, Plasmid Preparation, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Negative screen. One of the three cell types: 293T, 293T-ACE2 and 293T-ACE2-TMPRSS2 (30,000 cells) was co-transfected with plasmids encoding the T7 polymerase, luciferase and β-galactosidase reporter genes and then treated with 10 μM of individual drugs. Luciferase, β-galactosidase and the luciferase/β-galactosidase ratio are presented as ratios to the solvent controls which are set as 1. Data are presented as mean +/− SD of three repeats. * p < 0.05

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Fusion cell morphology. 50,000 293T-ACE2-TMPRSS2 pre-incubated with 10 μM of individual drugs were co-cultured with 50,000 donor 293T cells transfected with an empty vector or the SARS-CoV-2 spike protein (S2). 293T-ACE2 cells treated with tenofovir disoproxil fumarate under physiological pH was displayed at the bottom right corner; otherwise, all images represent fusion of 293T-ACE2-TMPRSS2 cells. Cells were fixed and stained with methylene blue. Bright-field images are of the same magnification x100 and scale. Fields of entire or mostly syncytia formation are framed with red squares. Fields of entire or moslty intact cells are framed with orange squares. Scattered syncytia are circled or outlined red and labelled S. Scattered intact cells are circled or outlined orange and labelled C. Sporadic intact cells and syncytia are marked by orange and red arrowheads, respectively. Drug images are from PubChem.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Incubation, Cell Culture, Transfection, Plasmid Preparation, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Rimonabant and anidulafungin inhibit ACE2-spike binding. Enzyme-linked immunosorbent assay of spike neutralizing antibody (S NAb) and drug inhibition of ACE2-spike binding. Data are presented as % binding of their respective solvent control and represent mean +/− SD of 2 repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Rimonabant, rucaparib, carvedilol and anidulafungin inhibit SARS-CoV-2 infection of TMPRSS2 cells. Mouse leukaemia virus pseudotyped with the spike protein (S) from severe acute respiratory syndrome coronavirus (SARS-1-S) and SARS-CoV-2 (SARS-2-S) was used to infect 293T-ACE2-TMPRSS2 cells, in a 96-well plate for 48 h in the presence of the drug, as indicated, with 1 h pre-treatment. Infectivity was measured as luciferase activity and expressed as % infectivity versus infected, solvent control. Viability was measured by XTT assays in un-infected samples and expressed as % viability versus solvent control. Data are presented as a heat map of the mean of three repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Infection, Luciferase, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Pan-coronaviral and pan-viral fusion inhibitors. Mouse leukaemia virus pseudotyped with glycoprotein from vesicular stomatitis virus (VSV-G) and spike protein (S) from severe acute respiratory syndrome coronavirus (SARS-1-S), SARS-CoV-2 (SARS-2-S) and Middle East respiratory syndrome coronavirus (MERS-S), was used to infect 293T-ACE2 (for SARS-1-S, SARS-2-S, VSV-G) or Huh-7 (for MERS-S) cells, in a 96-well plate for 48 h in the presence of the drug, as indicated, with 1 h pre-treatment. Infectivity was measured as luciferase activity and expressed as % infectivity versus infected, solvent control. Viability was measured by XTT assays in un-infected samples and expressed as % viability versus solvent control. Data are presented as a heat map of the mean of three repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Infection, Luciferase, Activity Assay

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Baseline ACE2 level, demographical, clinical characteristics of the Control group and the COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Association of ACE2 level with Age and Days of hospital admission the Control group and the COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Comparison of ACE2 level between Control and COVID-19 group in association with different clinical parameters.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Comparison, Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Association of ACE2 level with variable clinical parameters in COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Significance Assay