Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 1 In‐house and commercial ACE2 enzymatic immunoassay (EIA) results of pre‐COVID‐19 donor control sera, COVID‐19 convalescent patient, and vaccine recipient sera. (A, B) IgM EIA results of COVID‐19 convalescent sera classified based on severity. (C, D) IgG EIA results of COVID‐19 convalescent sera classified based on severity. (E, F) IgG EIA results of COVID‐19 vaccine recipients based on type of vaccine. Bars represent median and interquartile range. Intergroup comparisons of medians were performed using Dunn's multiple comparisons test. Ns: not significant; *p ≤0.05; ***p ≤0.001; ****p ≤0.0001. ACE2, angiotensin‐converting enzyme 2; COVID‐19, coronavirus disease 2019.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay, Control

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 2 Correlations between ACE2 IgG enzymatic immunoassay optical densities (OD) and surrogate neutralizing antibody levels of CoronaVac (A, B) and Comirnaty (C, D) cohorts using commercial and in‐house ACE2 peptides. Strength of correlation was assessed using Spearman's rank correlation. ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 3 Trends of ACE2 IgG optical densities (ODs) using in‐house (A) and commercial (B) peptides for vaccine recipients testing positive at Day 56 post‐first dose. Each line represents trend for individual recipients. SNV020, SNV027, and SNV058 are CoronaVac recipients. BNT007, BNT012, BNT032, BNT081, BNT090, and BNT092 are Comirnaty recipients. The second timepoint is either Day 21 (for Comirnaty recipients) or Day 28 (for CoronaVac recipients). ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques:

Journal: bioRxiv

Article Title: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

doi: 10.1101/2021.06.15.448419

Figure Lengend Snippet: A) C ells transfected with expression PS receptor plasmids, AXL or TIM-1, with or without 50 ng of ACE2 and infected 48 hours later with SARS-CoV-2 (MOI = 0.5). Viral loads were determined 24 hours following infection. B-C) PS receptors, TIM-1 (B) and AXL (C) , enhance rVSV-luciferase/Spike infection at low concentrations of ACE2 are transfected. D) Virus binding of cells transfected with PS receptor plasmids with or without 50 ng of ACE2. rVSV/Spike was bound to transfected cells at 48 hpi and bound virus was measured via RT-qPCR. E) Supernatants from SARS-CoV-2 infected (MOI = 0.5) transfected HEK 293T cells were titered 48 hpi on Vero E6-TMPRSS2 and TCID 50 calculated by Spearman-Karber equation. These studies were performed with transfection of 50 ng of ACE2 plasmid. F) HEK 293T cells transfected with expression PS receptor plasmids, TYRO3 or TIM-4, with or without 50 ng of ACE2 and infected 48 hours later with SARS-CoV-2 (MOI = 0.5). Viral loads were determined 24 hours following infection. Data shown are pooled from at least 3 independent experiments ( A, B, C, D, E, F ). Data represented as means ± SEM. Student’s t-test (A , E) and multiple t-test (B , C) , One-Way ANOVA with multiple comparisons (D&F) ; asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D AF933), goat anti-AXL (R&D154), goat anti-TIM-1 (R&D 1750), goat anti-Tyro3 (R&D AF859), goat anti-TIM-4 (R&D 2929), goat anti-MerTK (R&D AF891), rabbit anti-TMPRSS2 (Abcam ab92323).

Techniques: Transfection, Expressing, Infection, Luciferase, Binding Assay, Quantitative RT-PCR, Endpoint Dilution Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

doi: 10.1101/2021.06.15.448419

Figure Lengend Snippet: A-B) PS liposomes interfere with rVSV-luciferase/Spike infection. HEK 293T cells transfected with TIM-1 plasmid and 50 ng of ACE2 plasmid ( A ) or AXL plasmid and 50 ng of ACE2 plasmid ( B ) were infected with rVSV-luciferase/Spike in the presence of increasing concentrations of PS or PC liposomes and assessed for luciferase activity at 24 hours following infection. C) HEK 293T cells were transfected with WT or PS binding pocket mutant TIM-1 plasmids with or without 50 ng of ACE2 expressing plasmid and infected 48 hours later with rVSV-luciferase/Spike pseudovirions. Luminescence fold change were compared to mock transfected lysates that were set to a value of 1. D) AXL is unable to directly interact with purified, soluble SARS-CoV-2 spike/Fc. HEK 293T cells transfected with AXL or ACE2 were incubated with soluble spike protein (S1/S2)-Fc, S1 RBD-Fc or S1 NTD-Fc and subsequently incubated with an Alexa 647 secondary. Spike protein binding was detected by flow cytometry. E) AXL does not bind to the NTD of SARS-CoV-2 spike. Biolayer interferometry association curves show that immobilized AXL-Fc fails to interact with purified NTD of spike. Data are pooled from at least 3 independent experiments ( A, B ) or are representative of at least 3 experiments ( C, D, E ). Data represented as means ± SEM. Multiple t-test ( A, B ), One-way ANOVA with multiple comparisons ( C ); asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D AF933), goat anti-AXL (R&D154), goat anti-TIM-1 (R&D 1750), goat anti-Tyro3 (R&D AF859), goat anti-TIM-4 (R&D 2929), goat anti-MerTK (R&D AF891), rabbit anti-TMPRSS2 (Abcam ab92323).

Techniques: Luciferase, Infection, Transfection, Plasmid Preparation, Activity Assay, Binding Assay, Mutagenesis, Expressing, Purification, Incubation, Protein Binding, Flow Cytometry

Journal: bioRxiv

Article Title: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

doi: 10.1101/2021.06.15.448419

Figure Lengend Snippet: A) HEK 293T cells were transfected with ACE2 and TMPRSS2 as noted and infected at 48 h with VSV-luciferase/Spike. At 24 hpi, luminescence activity was determined. Findings are shown relative to empty vector (Mock) transfected cells. Panel depicts one representative experiment. Students t-tests. B) TMPRSS2 expression enhances rVSV-luciferase/Spike entry at low levels of ACE2 expression. HEK 293T cells were transfected as indicated and pseudovirion entry assessed by measuring luminescence activity at 24 hpi. C) Transfected HEK 293T cells were transfected and infected with VSV-luciferase/Spike at 48 h in the presence or absence of E-64 (300 μM). Luciferase activity was determined 24 hpi. Data are pooled from at least 3 independent experiments ( B, C ) or are representative of at least 3 experiments ( A ). Data represented as means ± SEM. Student’s T-tests (A) Multiple t-tests ( B ), Two-way ANOVA with row-wise multiple comparisons ( C ); asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D AF933), goat anti-AXL (R&D154), goat anti-TIM-1 (R&D 1750), goat anti-Tyro3 (R&D AF859), goat anti-TIM-4 (R&D 2929), goat anti-MerTK (R&D AF891), rabbit anti-TMPRSS2 (Abcam ab92323).

Techniques: Transfection, Infection, Luciferase, Activity Assay, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

doi: 10.1101/2021.06.15.448419

Figure Lengend Snippet: A) PS liposomes interfere with SARS-CoV-2 pseudovirion entry. Vero E6 cells were treated with PS or PC liposomes and incubated with VSV-GFP/Spike pseudovirions for 24 hours. Entry was detected by GFP fluorescence. B) PS liposomes disrupt SARS-CoV-2 binding. Vero E6 cells were incubated with SARS-CoV-2 (MOI = 5) at 10°C for 1 hour, washed extensively, and viral load assessed by RT-qPCR. C) AXL signaling inhibitor bemcentinib inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with bemcentinib and infected with SARS-CoV-2 (MOI = 0.01). Viral loads were measured 24 hpi by RT-qPCR. D) Bemcentinib inhibition of SARS-CoV-2 infection is most efficacious at early time points during infection. Vero E6 cells were challenged with SARS-CoV-2 (MOI = 0.01) and treated with either the vehicle control or 1 μM bemcentinib at the indicated time. Viral loads were measured 24 hpi by RT-qPCR. E) Vero E6 cells were treated with 1 μM bemcentinib, infected with SARS-CoV-2 (MOI = 0.01) and mRNA harvested 18 hpi. mRNA was deep sequenced on an Illumina platform, and viral loads were calculated by alignment to the SARS-CoV-2 genome. F) Broad spectrum TAM inhibitor BMS-777607 inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with inhibitor at indicated concentrations for 1 hour, challenged (MOI = 0.01), and viral loads measured 24 hpi by RT-qPCR G) STED micrographs showing staining for ACE2 (red) and AXL (green) and merged in Vero E6 cells. Insets are enlarged images from regions highlighted by yellow rectangles. White arrows indicate shared vesicular structures between the two channels. Yellow arrowheads indicate objects that are only seen in one channel. Plot profiles are shown in S4F , representing signal intensity along the yellow lines in the merged panels. H) Pearson’s correlation coefficients of ACE2 and AXL were calculated for n=20 mock and infected cells (ROI determined by cell borders). Data are pooled from at least 3 independent experiments ( B, D, F ) or are representative of at least 3 experiments ( A, C, G, H ). Data are represented as means ± SEM. Multiple t-tests ( A ) student’s t-test ( B, C, F, H ); asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D AF933), goat anti-AXL (R&D154), goat anti-TIM-1 (R&D 1750), goat anti-Tyro3 (R&D AF859), goat anti-TIM-4 (R&D 2929), goat anti-MerTK (R&D AF891), rabbit anti-TMPRSS2 (Abcam ab92323).

Techniques: Incubation, Fluorescence, Binding Assay, Quantitative RT-PCR, Infection, Inhibition, Staining

Journal: bioRxiv

Article Title: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

doi: 10.1101/2021.06.15.448419

Figure Lengend Snippet: A-F) SARS-CoV-2 infection is reduced by AXL inhibition in multiple human lung cell lines. In order: A549 ACE2 , H1650, HCC1944, H1819, HCC2302, Calu3 were treated with the indicated inhibitors for 1 hour and challenged with SARS-CoV-2 (MOI = 0.5) for 24 hours. Viral load was assessed by RT-qPCR. G) HCC2302 cells were treated with bemcentinib at the indicated concentrations for 1 hour and infected with SARS-CoV-2 (MOI = 0.5). Input virus was removed 6 hpi and supernatant was collected at 24 and 48 hpi and titered by TCID 50 assays on Vero E6-TMPRSS2 cells. TCID 50 /mL was calculated by the Spearmann-Karber method. H) A549 ACE2 were treated with bemcentinib as indicated, infected with SARS-CoV-2 (MOI = 0.5) and mRNA harvested 24 hpi. mRNA was sequenced, and viral loads calculated by alignment to the SARS-CoV-2 genome. Data are pooled from at least 3 independent experiments ( F ) or are representative of at least 3 experiments ( A, B, C, D, E, G ). Data represented as means ± SEM. Student’s t-test; asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D AF933), goat anti-AXL (R&D154), goat anti-TIM-1 (R&D 1750), goat anti-Tyro3 (R&D AF859), goat anti-TIM-4 (R&D 2929), goat anti-MerTK (R&D AF891), rabbit anti-TMPRSS2 (Abcam ab92323).

Techniques: Infection, Inhibition, Quantitative RT-PCR

Journal: Nature genetics

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

doi: 10.1038/s41588-022-01110-2

Figure Lengend Snippet: Fig. 3 | Secondary screens in Calu-3, Caco-2-ACE2, A549-ACE2 and Huh7.5.1-ACE2 cells. a, Schematic of secondary library design and screen strategy. b, Cumulative distribution plots analyzing overlap of top hits between primary and secondary screens. Putative hit genes from the primary screen are ranked by mean z-score, and classified as validated hits based on mean z-score in the secondary screen, using a threshold of greater than 3 for KO or less than −3 for activation. AUC, area under the curve. c, Heatmap comparison of top resistance and sensitization hits from secondary KO screens across cell lines. d, Heatmap comparison of top resistance and sensitization hits from secondary activation screens across cell lines.

Article Snippet: Cells were lysed in lysis buffer (10 mM Tris 1 M (pH 7.6), NaCl 150 mM, Triton X100 1%, EDTA 1 mM and deoxycholate 0.1%) supplemented with sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 100 mM DTT and 0.02% bromophenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies against ACE2 (ProteinTech 21115-1-P; diluted 1:1,000), AP1G1 (Bethyl Laboratories, A304-771A; 1:1,000), AP1B1 (Proteintech, 16932-1-AP; 1:500), AAGAB (Bethyl Laboratories, A305-593A; 1:1,000), or Actin (Sigma-Aldrich, A1978; 1:5,000), followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies (Thermo Fisher Scientific; 1:2,500), or using an anti-GAPDH antibody conjugated to horseradish peroxidase (Sigma-Aldrich, G9295; 1:5,000) and chemiluminescence Clarity or Clarity Max substrate (Bio-Rad).

Techniques: Activation Assay, Comparison

Journal: Plants

Article Title: Antcins from Antrodia cinnamomea and Antrodia salmonea Inhibit Angiotensin-Converting Enzyme 2 (ACE2) in Epithelial Cells: Can Be Potential Candidates for the Development of SARS-CoV-2 Prophylactic Agents

doi: 10.3390/plants10081736

Figure Lengend Snippet: Antcins reduced human ACE2 levels in HT-29 cells. HT-29 cells were incubated with the indicated concentration of antcins for 48 h. Cell lysates were subjected to determine ACE2 levels using a commercially available ELISA kit. Values represent the mean ± SD of three independent experiments. p -values of less than 0.01 **, and 0.001 *** were considered statistically significant for the antcins treatment group vs. the control group.

Article Snippet: Antibody against ACE2 was obtained from Arigo Biolaboratories (Hsinchu, Taiwan).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Plants

Article Title: Antcins from Antrodia cinnamomea and Antrodia salmonea Inhibit Angiotensin-Converting Enzyme 2 (ACE2) in Epithelial Cells: Can Be Potential Candidates for the Development of SARS-CoV-2 Prophylactic Agents

doi: 10.3390/plants10081736

Figure Lengend Snippet: Antcins inhibited ACE2 protein expression in HT-29 cells. ( A ) HT-29 cells were incubated with the indicated concentration of antcins for 48 h. Protein expression levels of ACE2 was determined by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. The relative density of one representative experiment is shown, where ACE2 signal was normalized with GAPDH signal. ( B , C ) Relative expression of ACE2 and TMPRSS2 mRNAs in HT-29 cells. Total RNA was extracted from cells treated with indicated concentration of antcins for 48 h. The transcription levels of ACE2 and TMPRSS2 were quantified by qPCR and a representative experiment is shown. The Δ ct values of ACE2 and TMPRSS2 mRNAs were normalized to GAPDH mRNA. Values represent the mean ± SD of two independent experiments. p -values of less than 0.05 *, 0.01 **, 0.001 ***, and 0.0001 **** were considered statistically significant for the antcins treatment group vs. the control group.

Article Snippet: Antibody against ACE2 was obtained from Arigo Biolaboratories (Hsinchu, Taiwan).

Techniques: Expressing, Incubation, Concentration Assay, Western Blot

Journal: Biomedicine & Pharmacotherapy

Article Title: GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies

doi: 10.1016/j.biopha.2020.110816

Figure Lengend Snippet: Effect of GB-2 on ACE2 and TMPRSS2 expression. (A, B) Total cell extracts of HepG2 cells (A) or 293 T cells (B) were harvested from untreated cells and cells treated with GB-2 for 24 h. The protein was immunoblotted with polyclonal antibodies specific for ACE2 or TMPRSS2. GAPDH was used as an internal loading control. (C, D) Total mRNA was extracted from the HepG2 cells (C) or 293 T cells (D) after treat with GB-2 for 24 h. The coding regions of human ACE2 were used as probes for real time polymerase chain reaction analysis. All the results are representative of at least three independent experiments. (Error bars = mean ± S.E.M. Asterisks (*) mark samples significantly different from control group with p < 0.05).

Article Snippet: These primary antibodies included: anti-ACE2 antibody (Cell Signaling, ratio: 1:1000), anti-TMPRSS2 antibody (Abcam, ratio: 1:1000) and anti-GAPDH antibody (Santa Cruz, IB: 1:10000).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Biomedicine & Pharmacotherapy

Article Title: GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies

doi: 10.1016/j.biopha.2020.110816

Figure Lengend Snippet: Effect of GB-2 on ACE2 and TMPRSS2 expression on mouse model. (A, C) Representative IHC staining photomicrographs of the lung (A) and kidney (C) tissue in mice. (B, D) Quantitative results of IHC staining, which were presented as IOD/area and were proportional to the levels of ACE2 or TMPRSS2 in the lung (B) and kidney (D) tissue. (n = 5 per group, Error bars = mean ± S.E.M. Asterisks (*) mark samples significantly different from control group with p < 0.05).

Article Snippet: These primary antibodies included: anti-ACE2 antibody (Cell Signaling, ratio: 1:1000), anti-TMPRSS2 antibody (Abcam, ratio: 1:1000) and anti-GAPDH antibody (Santa Cruz, IB: 1:10000).

Techniques: Expressing, Immunohistochemistry, Control

Journal: Biomedicine & Pharmacotherapy

Article Title: GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies

doi: 10.1016/j.biopha.2020.110816

Figure Lengend Snippet: Effect of theaflavin on ACE2 and TMPRSS2 expression. (A) The structure of theaflavin. (B) 293 T cells were measured by XTT assay after indicated hours of culturing in the presence of theaflavin. (C) Total cell extracts of 293 T cells were harvested from untreated cells and cells treated with theaflavin for 24 h. The protein was immunoblotted with polyclonal antibodies specific for ACE2 or TMPRSS2. GAPDH was used as an internal loading control. (D) Total mRNA was extracted from the 293 T cells after treat with theaflavin for 24 h. The coding regions of human ACE2 were used as probes for real time polymerase chain reaction analysis. All the results are representative of at least three independent experiments. (Error bars = mean ± S.E.M. Asterisks (*) mark samples significantly different from control group with p < 0.05).

Article Snippet: These primary antibodies included: anti-ACE2 antibody (Cell Signaling, ratio: 1:1000), anti-TMPRSS2 antibody (Abcam, ratio: 1:1000) and anti-GAPDH antibody (Santa Cruz, IB: 1:10000).

Techniques: Expressing, XTT Assay, Control, Real-time Polymerase Chain Reaction