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: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Phylogenetic trees depicting N genes isolated from nose-throat swabs by SGS. IDs of individuals from whom swabs were isolated are indicated in the figure. Black rectangles depict the dominant N gene isolated from each swab sample. Red rectangles depict N genes with missense mutations, grey rectangles depict N genes with silent mutations and blue rectangles depict N genes with nonsense mutations. Mutations that are not documented in the GSAID are marked with an asterisk. For each N-gene variant, the nucleotide substitution is indicated (based on Wuhan-Hu-1 numbering). The segment with the number below each phylogenetic tree shows the length of branch that represents an amount genetic change. The amount of genetic change is the number of nucleotide substitutions divided by the length of the N-gene sequence. (B) Pie charts showing the proportion of all N-gene sequences isolated from each swab sample. The White pie slice depicts unmutated sequences. The Colored pie slices depict mutated N genes. The different mutations are indicated in the figure. The number in the middle of the pie chart depicts the total number of N-gene variants that were sequenced. (C) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The x-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the y-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated N proteins and unmutated (Wuhan-Hu-1) N protein (WT). The x-axis depicts the mutation in the N proteins of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Isolation, Variant Assay, Sequencing, Produced, Infection, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Schematic representation of the ten spike variants that were tested for infectivity. Amino acids substitution in each spike mutants is shown in the figure. NTD = N-terminal domain, RBD = receptor binding domain, FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain, CT = cytoplasmic domain. (B) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The y-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the x-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. (C) FACS staining of 293T-ACE2 cells. The gray histogram shows the staining of the 293T-ACE2 cells with secondary antibody only. The empty black histograms depict the staining anti-ACE2 antibody or RBD-Ig. Shown is one representative experiment out of three preformed. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated spikes, unmutated (Wuhan-Hu-1) spike protein and bald pseudovirus. The x-axis depicts the mutation in the spike of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, *p < 0.05, **p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Infection, Binding Assay, Produced, Staining, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A, C) OD values of ELISA against spike S1 subunit of plasma from vaccinated individuals (A) and of convalescent plasma (C). Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. OD values of control plasma (unvaccinated healthy individual) are shown in the red dashed graph. (B) The luminescence values derived from 293T-ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with increasing concentrations of plasma from vaccinated individuals. The plasma dilution is shown in the x-axis. Luminescence values after incubation with control (unvaccinated healthy individual) is shown in the red-dashed graph. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown. (D) The luminescence values derived from 293T-ACE2 cells 48 hours post infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with convalescent plasma at 10 −3 dilution. The study ID of the plasma samples is shown in the x-axis. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown.

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Infection, Expressing, Incubation

Journal: Autophagy

Article Title: Autophagy and post-ischemic conditioning in retinal ischemia

doi: 10.1080/15548627.2020.1767371

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: p-RPS6KB1 (Ser421) , Bioss Antibodies , Rabbit Polyclonal , BS-6421RA555 , 1:500.

Techniques:

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Immunodetection of ACE2. ( a ) Vero or A549 cells were incubated with the indicated anti-ACE2 antibodies prior to fixation. Right panels show the background of the secondary anti-rabbit IgG antibody. Insets show the cell contours. ( b ) A549 cells cultured for 5 days or 7 days after plating were stained with the indicated anti-ACE2 antibodies after fixation with 4% (w/v) PFA and permeabilization with 0.1% (v/v) Triton X-100 for 5 min. Nuclei were counterstained with DAPI. (p, rabbit polyclonal; ab, rabbit polyclonal antibody from Abcam). Bars, 20 μm. ( c ) Detection of ACE2 by western blot. Total cell lysates from the indicated cell types containing 30 μg of protein, or 100 ng of purified ACE2 protein (ACE2-His) were analyzed by SDS-PAGE followed by immunoblot with the indicated anti-ACE2 antibodies. Left panels show a long and a short exposure of the blot of cell lysates and recombinant protein, respectively. Middle panels show a short exposure in both cases. In the right panel, the total protein on blots was visualized by staining with Simply Blue. ( d ) Scheme depicting the sequence of ACE2, the location of the epitopes of the antibodies used and the region spanned by the recombinant protein. SP, signal peptide; TM, transmembrane domain.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Immunodetection, Incubation, Cell Culture, Staining, Western Blot, Purification, SDS Page, Recombinant, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike constructs and ACE2 in several cell types employing different sequences for immunodetection. ( a ) Scheme of the incubation and washing steps performed for immunodetection. Incubations were carried out for 1 h in the cold. After each incubation coverslips were washed three times with 200 μl of cold PBS. At the end of the procedure, an additional washing step with water was performed before coverslips were allowed to dry and and mounted. ( b ) Representative images from the detection of Spike constructs and ACE2 in the indicated cell lines employing the different immunodetection sequences. The graph shows the colocalization between Spike S1-Fc and ACE2 fluorescent signals for every cell type and immunodetection sequence assayed. Colocalization is expressed as the proportion of Spike S1 colocalizing with ACE2 signal (Manders’ coefficient), measured applying the automatic Costes’ threshold (n ≥ 8 per condition). Results are shown as mean values ± SEM; ns, non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 by ANOVA with Tukey’s post-test. Bars, 20 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Construct, Immunodetection, Incubation, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of vimentin and ACE2 in A549 cells. ( a ) Live A549 cells were incubated simultaneously with anti-vimentin and anti-ACE2 antibodies, after which they were incubated with a combination of the corresponding secondary antibodies prior to fixation. An illustrative image is depicted showing points of colocalization at intercellular contacts at mid-cell height, highlighted in the colocalization mask. In addition, an overlay of the region of interest delimited by the dotted square with the bright field image is shown to illustrate the juxtanuclear position of one of the colocalization points (white arrow); the contour of the nucleus is highlighted in magenta. The far right panel shows the top section of the region of interest along with the orthogonal projections centered in the colocalization point marked with the arrow. Bars, 20 μm. ( b – d ) Cells were fixed and permeabilized as specified below before immunodetection. ( b ) A549 cells were fixed with 4% (w/v) PFA and permeabilized with 0.1% (v/v) Triton for 5 min for staining with monoclonal antibodies against acetylated tubulin (acTubulin, left panels) or ARL13B (right panels). Nuclei were stained with DAPI. ( c ) A549 cells fixed and permeabilized as in ( b ), were stained with anti-ACE2 and anti-acetylated tubulin antibodies. ( d ) A549 cells were fixed with 2% (w/v) PFA, permeabilized with 0.1% (v/v) Triton X-100 and stained with anti-vimentin antibodies (SP20 or C-end) and either anti-acetylated tubulin or anti-ARL13B antibodies, as indicated. Images in ( b – d ) are single sections. In each case, the regions of interest (dotted square) are enlarged at the right. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Immunodetection, Staining

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike S1, ACE2 and vimentin at primary cilia. A549 cells were incubated with Spike S1-Fc at 37 °C for 90 min in complete medium. Non-permeabilized cells were stained with ( a ) anti-ACE2 and anti-vimentin 84-1 antibodies, ( b ) anti-ACE2 and anti-acetylated tubulin or ( c ) anti-vimentin SP20 and anti-ARL13B antibodies, followed by the corresponding secondary antibodies, and finally with anti-human IgG antibody to detect the Spike S1 Fc tag, before fixation. In ( a , b ) cells were fixed with 4% (w/v) PFA and in ( c ) with 2% (w/v) PFA. Images shown are single sections for each channel obtained by confocal microscopy, at middle height of the cells, and the corresponding overlays. Regions of interest (dotted squares) are enlarged in the lower panels for each condition. Colocalization masks for the signals of ( a ) vimentin and Spike, ( b ) ACE2 and Spike, and ( c ) vimentin and Spike, are shown at the right as white signals on black background; numbers in insets correspond to the Pearson’s coefficient and the percentage of colocalization for the regions shown. Colocalization analysis was performed with Leica software. Arrows in c point to structures showing colocalization. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Staining, Confocal Microscopy, Software

Journal: Gastroenterology

Article Title: Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

doi: 10.1053/j.gastro.2020.09.042

Figure Lengend Snippet: sEVs and exomeres containing ACE2 can bind SARS-CoV-2 through the virus spike (S) protein. ( A ) Immunoblot analysis of cells and sEVs purified by high-resolution density gradients. ( B ) LIM1215 ( left ) and DiFi ( right ) samples were fractionated followed by immunoblotting. Arrow , full-length ACE2; ∗Ectodomain glycoform 1 of ACE2; ∗∗Ectodomain glycoform 2 of ACE2; ACE2 (ecto), ectodomain-specific monoclonal antibody; ACE2 (intra), intracellular-specific monoclonal antibody; I, glycosylated DPP4; II, nonglycosylated DPP4; anti-CD63 and anti-HSPA13 are used as exosomal and exomere markers, respectively; NV, nonvesicular; Exom, exomere. ( C ) Fluorescence-activated vesicle sorting analysis for ACE2 expression in sEV pellets ( P ) and exomeres from DiFi cells using an ectodomain-specific polyclonal antibody directly conjugated to PE. ( D ) Immunoblot analysis of inducible nitric oxide synthase (iNOS) and ACE2 expression in LIM1215 cells treated with indicated doses of interferon gamma (INF-γ) and TNF-α. ( E ) Sample inputs from DiFi cells ( left ) and agarose-conjugated Sambucus nigra agglutinin (SNA) lectin pull-downs ( right ) were analyzed by immunoblotting. β1-Integrin and epidermal growth factor receptor (EGFR) are known to be α2,6-sialylated and were used as positive controls. ( F ) Binding of human recombinant soluble ACE2 to the RBD of SARS-CoV-2 S protein. IP, immunoprecipitation; IB, immunoblotting. ( G ) Binding of DiFi exomeres with RBD of SARS-CoV-2 S protein. ( H ) Binding of DiFi cells, sEV-P, and exomeres with the S1 subunit of SARS-CoV-2 S protein. ( I ) Fluorescence-activated vesicle sorting analysis for binding of DiFi sEV-P and exomere to a mouse Fc-tagged RBD of SARS-CoV-2 S protein. ( J ) Schematic illustration of ( i ) SARS-CoV-2 viral infection of ACE2-positive cells and ( ii ) inhibition of infection due to hypothetical viral decoy binding by ACE2-positive sEVs and exomeres. Similarly, DPP4-positive sEVs and exomeres may act as decoys for MERS-CoV.

Article Snippet: Primary antibodies were as follows: anti-ACE2 (Bioss, bs-1004R-PE, PE-conjugated), anti-ACE2 (Abcam, ab108252), anti-ACE2 (R&D, AF933), and SARS-CoV-2 mouse Fc fusion (mouse, Crowe Laboratory).

Techniques: Western Blot, Purification, Fluorescence, Expressing, Binding Assay, Recombinant, Immunoprecipitation, Infection, Inhibition

Journal: Gastroenterology

Article Title: Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

doi: 10.1053/j.gastro.2020.09.042

Figure Lengend Snippet: ( A ) Immunoblot analysis of colorectal cancer cells with indicated antibodies. ( B ) Immunoblot analysis of DKO-1 cells, sEV, and nonvesicular (NV) samples with indicated antibodies. ( C ) Fluorescence-activated vesicle sorting analysis for ACE2 expression in DiFi sEV-P using an ectodomain-specific monoclonal ACE2 primary antibody followed by an Alexa-647 secondary antibody. ß-actin serves as a loading control.

Article Snippet: Primary antibodies were as follows: anti-ACE2 (Bioss, bs-1004R-PE, PE-conjugated), anti-ACE2 (Abcam, ab108252), anti-ACE2 (R&D, AF933), and SARS-CoV-2 mouse Fc fusion (mouse, Crowe Laboratory).

Techniques: Western Blot, Fluorescence, Expressing

Journal: Gastroenterology

Article Title: Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

doi: 10.1053/j.gastro.2020.09.042

Figure Lengend Snippet: ( A ) Immunoblot analysis of inducible nitric oxide synthase (iNOS) and ACE2 expression in cells cultured under standard conditions for EV and exomere isolation. ( B ) Immunoblot analysis of iNOS and ACE2 expression in cells treated with indicated concentrations of interferon gamma (INF-γ) and TNF-α. ( C ) Immunoblot analysis for ACE2 from LS174T cells, sEV-Ps, and Exom. ( D ) Fluorescence-activated vesicle sorting analysis of LS174T, sEV-Ps, and exomeres binding to a mouse Fc-tagged RBD of SARS-CoV-2 spike protein S1. Actin serves as a loading control.

Article Snippet: Primary antibodies were as follows: anti-ACE2 (Bioss, bs-1004R-PE, PE-conjugated), anti-ACE2 (Abcam, ab108252), anti-ACE2 (R&D, AF933), and SARS-CoV-2 mouse Fc fusion (mouse, Crowe Laboratory).

Techniques: Western Blot, Expressing, Cell Culture, Isolation, Fluorescence, Binding Assay

Journal: Oncology Reports

Article Title: Combination of gemcitabine and erlotinib inhibits recurrent pancreatic cancer growth in mice via the JAK-STAT pathway

doi: 10.3892/or.2018.6198

Figure Lengend Snippet: Gemcitabine in combination with erlotinib in recurrent tumors of BxPC-3 cells induces increased activity of caspase-3 and caspase-9. (A and B) Immunohistochemical staining of active caspase-9 and active caspase-3 in tumor tissue sections revealed active caspase-9 activation by combination treatment and active-caspase-3 activation by either monotherapy and by combination treatment in the BxPC-3 cells. Lower panels displayed higher magnification (original magnification, ×400) of the boxed areas in the upper panels. Scale bars, 10 µm. (C and D) Visual fields ( – ) in each section of irradiation were photographed. The staining intensity and the percentage of positive cells were calculated and scored in each section. The total scores of each visual field were first analyzed using the homogeneity of variance test, followed by the change of variable test. The scores were presented as the mean ± SD. GraphPad Prism 5 was used to plot cartograms. *P<0.05 denotes the controls vs. the treatment groups. # P<0.05 denotes the monotherapy (E or G) vs. the combination (E+G) groups.

Article Snippet: The following primary antibodies were used: anti-caspase-9 (rabbit polyclonal; bs-0049R) and anti-caspase-3 (rabbit polyclonal; bs-0081R), both purchased from BIOSS (Beijing, China).

Techniques: Activity Assay, Immunohistochemical staining, Staining, Activation Assay, Irradiation

Journal: Oncology Reports

Article Title: Combination of gemcitabine and erlotinib inhibits recurrent pancreatic cancer growth in mice via the JAK-STAT pathway

doi: 10.3892/or.2018.6198

Figure Lengend Snippet: Gemcitabine in combination with erlotinib in recurrent tumors of PANC-1 cells induces increased activity of caspase-3 and caspase-9. (A and B) Immunohistochemical staining of active caspase-9 and active caspase-3 in tumor tissue sections revealed active caspase-9 activation by either the monotherapy and by the combination treatment and active-caspase-3 activation by erlotinib monotherapy and by the combination treatment in PANC-1 cells. Lower panels displayed higher magnification (original magnification, ×400) of the boxed areas in the upper panels. Scale bars, 10 µm. (C and D) Visual fields ( – ) in each section of irradiation were photographed. The staining intensity and the percentage of positive cells were calculated and scored in each section. The total scores in each visual field were first analyzed using the homogeneity of variance test, followed by the change of variable test. The scores were presented as the mean ± SD. GraphPad Prism 5 was used to plot cartograms. *P<0.05 indicates the controls vs. the treatment groups. # P<0.05 indicates the monotherapy (E or G) vs. the combination (E+G) groups.

Article Snippet: The following primary antibodies were used: anti-caspase-9 (rabbit polyclonal; bs-0049R) and anti-caspase-3 (rabbit polyclonal; bs-0081R), both purchased from BIOSS (Beijing, China).

Techniques: Activity Assay, Immunohistochemical staining, Staining, Activation Assay, Irradiation