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: Briefly, cells were staining with Fixable Viability Dye eFluor 780 (eBiosciences), goat anti-human ACE2 (R&D AF933) followed by secondary were donkey anti-goat IgG (H+L) Alexa Fluor 647 (Jackson Immuno Research; Ref# 705-605-003) To measure the intracellular expression of hACE2, cells were surface stained with Fixable Viability Dye eFluor 780, fixed (PFA 4%), permeabilized (1X PBS + 0.5%Tween20) and stained intracellularly using goat anti-human ACE2 (R&D AF933) followed by secondary donkey anti-goat labeled with AF647.

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: Briefly, cells were staining with Fixable Viability Dye eFluor 780 (eBiosciences), goat anti-human ACE2 (R&D AF933) followed by secondary were donkey anti-goat IgG (H+L) Alexa Fluor 647 (Jackson Immuno Research; Ref# 705-605-003) To measure the intracellular expression of hACE2, cells were surface stained with Fixable Viability Dye eFluor 780, fixed (PFA 4%), permeabilized (1X PBS + 0.5%Tween20) and stained intracellularly using goat anti-human ACE2 (R&D AF933) followed by secondary donkey anti-goat labeled with AF647.

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: Briefly, cells were staining with Fixable Viability Dye eFluor 780 (eBiosciences), goat anti-human ACE2 (R&D AF933) followed by secondary were donkey anti-goat IgG (H+L) Alexa Fluor 647 (Jackson Immuno Research; Ref# 705-605-003) To measure the intracellular expression of hACE2, cells were surface stained with Fixable Viability Dye eFluor 780, fixed (PFA 4%), permeabilized (1X PBS + 0.5%Tween20) and stained intracellularly using goat anti-human ACE2 (R&D AF933) followed by secondary donkey anti-goat labeled with AF647.

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 , 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: Briefly, cells were staining with Fixable Viability Dye eFluor 780 (eBiosciences), goat anti-human ACE2 (R&D AF933) followed by secondary were donkey anti-goat IgG (H+L) Alexa Fluor 647 (Jackson Immuno Research; Ref# 705-605-003) To measure the intracellular expression of hACE2, cells were surface stained with Fixable Viability Dye eFluor 780, fixed (PFA 4%), permeabilized (1X PBS + 0.5%Tween20) and stained intracellularly using goat anti-human ACE2 (R&D AF933) followed by secondary donkey anti-goat labeled with AF647.

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: Briefly, cells were staining with Fixable Viability Dye eFluor 780 (eBiosciences), goat anti-human ACE2 (R&D AF933) followed by secondary were donkey anti-goat IgG (H+L) Alexa Fluor 647 (Jackson Immuno Research; Ref# 705-605-003) To measure the intracellular expression of hACE2, cells were surface stained with Fixable Viability Dye eFluor 780, fixed (PFA 4%), permeabilized (1X PBS + 0.5%Tween20) and stained intracellularly using goat anti-human ACE2 (R&D AF933) followed by secondary donkey anti-goat labeled with AF647.

Techniques: Infection, Inhibition, Quantitative RT-PCR

Journal: eLife

Article Title: ACE2: Evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities

doi: 10.7554/eLife.61390

Figure Lengend Snippet: Available validation data for the antibodies used in the studies described in this review in accordance to the pillars defined by the Antibodypedia validation initiative ( Uhlen et al., 2016 ). Provider refers to the information found in the website of the company. IB: Immunoblot. IHC: Immunohistochemistry. IF: Immunofluorescence. Enhanced validation, Supportive validation, No data available, + Positive detection, +/- Weak detection, - Absence of detection.

Article Snippet: AF933 Immunogen: 18-74aa , IB , , + Ovary, testis and kidney , + Airway and distal lung, ALI-cultured airway epithelial cells (correlation with mRNA levels), Calu-3 and Caco-2 cells ( ; ) - A549 (expected), Huh-7 cells ( ; ) .

Techniques: Biomarker Discovery, Western Blot, Immunohistochemistry, Immunofluorescence, Immunohistochemistry-IF, Transfection, Staining