Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: Immunohistochemistry for ACE2 in human pancreatic tissue sections (case #110118) using R&D MAB933 antibody. ACE2 is markedly expressed in microvasculature associated cells (panel-a and -b), in some rare ductal cells (panel-c and -d) and in a subset of endocrine cells within pancreatic islets (panel-e and -f). Scale bars in panel-a, -c and -e: 150 μm. Scale bars in panel-b, -d and-f: 70 μm. Zoom-in images are reported in panel -b, -d and -f

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: ( A ) Representative image of human pancreatic Formalin-Fixed Paraffin Embedded (FFPE) section stained for ACE2 in case #301118. In panel-a, a representative image of a pancreatic section showing two adjacent lobules (blue and red dotted lines) with different staining for ACE2 in endothelial cells/pericytes. A specific segmentation of the two lobules with high (blue) (zoom-in, panel-b) and low or null expression of ACE2 (red) (zoom-in, panel-c) is shown, suggesting lobularity of ACE2 expression in exocrine endothelial cells/pericytes of human pancreas. Scale bar in panel-a: 100 μm. Scale bar in panel-b and -c: 30 μm. ( B ) Double immunofluorescence staining of ACE2 (green) and CD31 (red) in FFPE pancreas sections from Body01A of Case #110118 (panel-a to −d) and of Body01B of Case #141117 (panel-e to −i). Digital zoom-in overlay images are shown in panels -d, -h and −i. Scale bar in panel-d and −g: 100 μm.

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Double Immunofluorescence Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: ( A ) Aligned sequences and structures of recently described ACE2 isoforms, long-ACE2 (805aa, ~110 kDa) and short-ACE2 (459aa, ~50kDa). Main ACE2 protein domains are reported with different colours. ( B ) R&D MAB933, ( C ) Abcam Ab15348 and ( D ) Ab108252 antibody predicted target sequence within the two ACE2 isoforms, alongside with immunohistochemistry staining distribution in pancreatic islets. Scale bars in (B), (C) and (D) are 100μm.

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Sequencing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: Triple immunofluorescence staining and image analysis of FFPE human pancreatic section stained for insulin (red), glucagon (blue) and ACE2 (green). ( A ) Representative islets of two different cases. Panel-a to -g: representative pancreatic islet of FFPE pancreas block Body01A of case #110118. Panel-h to -m: representative pancreatic islet of FFPE pancreas block Body01B of case #141117. Panel-e to -g: digital zoom in images of the pancreatic islet shown in panel-d. Panel-l and -m: digital zoom in images of the pancreatic islet shown in panel-k. Scale bar in panel-d and -k: 100 μm. ( B ) Colocalization rate analysis of overlapping ACE2-insulin and ACE2-glucagon in 128 single pancreatic islets of 7 different cases. p-value was calculated using Wilcoxon matched-pairs signed rank test. On the right: colocalization rate analysis of ACE2-insulin and ACE2-glucagon in each of the 7 cases analysed. For each case, a total of 7-11 islets/section were analysed. ( C ) Analysis of the intensity of ACE2 islet-related signals in the cases analyzed. Values are shown as fluorescence intensity of each islet reported as the sum of gray-scale values for each pixel normalized for the islets area (ROI, μm 2 ).

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Immunofluorescence, Staining, Blocking Assay, Fluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: Western Blot and Immunofluorescence analysis of EndoC-βH1 using ( A ) R&D monoclonal MAB933, ( B ) Abcam polyclonal Ab15348 and ( C ) Abcam monoclonal Ab108252 anti-ACE2 antibody. For each antibody adopted, specific target sequence is reported within the aligned ACE2 isoforms. In western blot analysis, molecular weight markers (from 15 kDa to 250 kDa) are reported; red arrows indicate long-ACE2 isoform (expected band of ~110kDa), while blue arrows indicate short-ACE2 isoform (~50kDa). ( D ) ACE2 (R&D MAB933) and insulin double immunofluorescence analysis in EndoC-βH1 cultured cells. Negative isotype primary antibody control (relative to ACE2 primary antibody) is shown in panel-a. Insulin (red) and ACE2 (green) are reported in panel-b and -c, while overlay is reported in panel-d. Digital zoom-in images are reported from panel-e to -h. Scale bar in panel -h = 15 μm.

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Western Blot, Immunofluorescence, Sequencing, Molecular Weight, Cell Culture, Control

Journal: bioRxiv

Article Title: SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature

doi: 10.1101/2020.07.23.208041

Figure Lengend Snippet: ( A-D ) ACE2 raw Ct values results in lung tissue (n=1, in duplicate), in enzymatic-isolated human pancreatic islets samples (n=4), in LCM-microdissected islets (n=5) and in EndoC-βH1 (n=4). ( E-G ) ACE2 expression values normalized using GAPDH and β2-microglobulin of the samples analysed in A-D. Values are reported as 2 -dCt . Mean ± S.D. values are shown.

Article Snippet: Cytokines-treated or untreated cells were fixed in 4% PFA for 10 min, washed for 10 min in 0.1 mol/L glycine, permeabilized in 0,25% Triton-X-100 for 5 min and blocked in 3% BSA+0.05% Triton-X100 in PBS without Ca 2+ and Mg 2+ for 30 min. EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat. A21435 - Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca 2+ and Mg 2+ and with monoclonal mouse anti-Human ACE2 (cat. MAB933, R&D System, Minneapolis, MS, USA) diluted 1:33 in BSA 1% in PBS without Ca 2+ and Mg 2+ for 1h at RT or with negative isotype control mouse IgG2a (cat. X0943 - Agilent Technologies, Santa Clara, CA, USA).

Techniques: Isolation, 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) 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: bioRxiv

Article Title: The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

doi: 10.1101/2024.08.08.606661

Figure Lengend Snippet: (A) K18-hACE2 mice were infected with SARS-CoV-2 WA1 strain and dosed with nirmatrelvir (300 mg/kg) or vehicle BID. Each group was composed of n=5 mice. The percent weight loss is presented as mean ± SD. (B) Survival curve in vehicle or nirmatrelvir treated mice based on the weight loss humane endpoint.

Article Snippet: A549-ACE2h cells were generated by stably expressing hACE2 ( ) and further selecting for high ACE2 expression levels via FACS with Alexa Fluor® 647 conjugated to a hACE2-specific antibody (FAB9332R, R&D systems).

Techniques: Infection

Journal: bioRxiv

Article Title: The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

doi: 10.1101/2024.08.08.606661

Figure Lengend Snippet: (A) K18-hACE2 mice were intranasally infected and intraperitoneally dosed as indicated with either vehicle (n=10) or AVI-4206 (n=15). Mice infected with WA1 N40D mutant, which lacks Mac1 catalytic activity, served as a positive control (n=10). Lungs were harvested at indicated time points for virus titration by plaque assay. (B) The percent body weight loss for all animals. The data are presented as mean ± SD. (C) Survival curve plotted based on the percent weight loss humane endpoint. (D) Viral load in the lungs and brain of infected mice at the indicated time points. The data are shown as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by Mann Whitney’s test relative to the vehicle control. (E) Schematics and graphs demonstrating the abundance of indicated cytokines at 4 and 7 days post-infection in the lungs of infected mice. The data are presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by two-tailed Student’s t-test relative to the vehicle control at each timepoint. None of the mice reached the humane endpoint at day 4 post-infection. For mice that reached the humane endpoint before day 7 post-infection, the tissues were collected and analyzed with mice at the 7 day time point.

Article Snippet: A549-ACE2h cells were generated by stably expressing hACE2 ( ) and further selecting for high ACE2 expression levels via FACS with Alexa Fluor® 647 conjugated to a hACE2-specific antibody (FAB9332R, R&D systems).

Techniques: Infection, Mutagenesis, Activity Assay, Positive Control, Virus, Titration, Plaque Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

doi: 10.1101/2024.08.08.606661

Figure Lengend Snippet: (A) K18-hACE2 mice were intranasally infected SARS-CoV-2 WA1 or SARS-CoV-2 WA1 Mac1 N40D mutant. Mice were treated as indicated with AVI-4206 (BID, 30 mg/kg) or vehicle. Each group was composed of n=10 mice (5 mice per time point). (B) The percent body weight loss is presented as mean ± SD. (C) Survival curve based on the percent body weight loss humane endpoint. (D) Viral load in the lung at indicated time points is presented as mean ± s.e.m. **, P < 0.01 by Mann Whitney’s test relative to the vehicle control.

Article Snippet: A549-ACE2h cells were generated by stably expressing hACE2 ( ) and further selecting for high ACE2 expression levels via FACS with Alexa Fluor® 647 conjugated to a hACE2-specific antibody (FAB9332R, R&D systems).

Techniques: Infection, Mutagenesis, Control

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: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in human fetal hippocampus. A Image of a coronal section of the temporal lobe showing the hippocampal formation. Insert represents the picture showed in ( B ). B Picture showing the Hippocampus Fimbrial Angle (HFA) between the fimbria and the Ammon’s horn (CA) ventricular surfaces. Inserts represent the areas that are showed in C, D, and F. ACE2 immunopositive (ACE2 +) migratory cells were observed from the HFA ventricular epithelium (VE) to the subpial region (pial surface: ps), through a radial migratory stream of cells toward the dentate gyrus (DGMS). C High power photomicrograph of the HFA showing the distribution of ACE2 + cells in the ventricular epithelium (VE) and the subventricular zone (SVZ). D High power photomicrograph of ventricular surface showing ACE2 + cells in VE and the subventricular zone (SVZ) of proximal ( D ) side of the HFA. E Ventricular surface showing ACE2 immunopositive particles accumulate on the basal pole membrane of VE progenitors (arrows). F Picture showing ACE2 + migrating cells reaching the developing Dentate Gyrus (DG) at the pial surface (ps) of FDS. G High power photomicrographs of migratory ACE2 + cells reaching he ps of the DGMS. H – J High power pictures showing ACE2 expression in the advance processes of migrating cells: (arrows in J and H ), and cells around blood vessels (arrows in I ). K – M ACE2 expression in choroidal plexus cells. Both epithelial cells (ec) in the ventricular surface, and stromal pericytes (pc) around blood vessels (bv) were immunopositive (arrows). N ACE2 expression in DG ventricular epithelium and DGMS at the caudal pole of hippocampus (FC and IG). Scale bar: A 500 µm; B 300 µm; C , D 50 µm; E 10 µm; F 150 µm; G , N 100 µm; H–K 15 µm; L , M 5 µm. bv blood vessel, CA cornu ammonis, DGMS dentate gyrus migratory stream, ec epithelial cell, FDS fimbrio-dentate sulcus, Fi fimbria, HFA hippocampus fimbrial angle, pc pericytes, ps pial surface, SVZ sub-ventricular zone, st choroidal plexus stroma, VE ventricular Epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Membrane

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: Migration routes of DG progenitors. A Picture of a coronal section of the temporal lobe showing GFAP expression in radial glia cells and fibers of the Sub-ventricular Zone (SVZ) and Migratory Stream (DGMS) of the Dentate Gyrus (DG), from the Hippocampus Fimbrial Angle (HFA) to the pial surface (ps). DGMS has been delimited by arrows. B High power picture of the DG Ventricular Epithelium (VE), Sub-ventricular Zone (SVZ) and intermediate zone (IZ). Arrows label GFAP positive fibers. C High power picture of ACE2-expressing cells (cells with DAB-nickel precipitate; arrows) following GFAP radial glia fibers of DG ventricular epithelium (VE; DAB precipitate; arrows head). D , E Cresyl violet staining showing the DG ventricular (VE) and sub-ventricular (SVZ) zones with perivascular accumulation of cells crossing the sub-ventricular withe matter (alveus) in the DG migratory stream (DGMS; arrows). F ACE2 expression in perivascular migrating cells (arrowheads) and pericytes (arrows) in the alveus. G Doublecortin expression in DG migrating neuronal precursors and neurons in DG molecular layer (DGML). ACE2 is expressed in migratory precursors of the DGMS (DAB-nickel precipitate; arrows) but not in DGML, single doublecortin expressing cells (DAB precipitate). Scale bar: A 200 µm; B 100 µm; C – E 50 µm; F 25 µm. bv blood vessel, DGH Dentate Gyrus Hilux, DGMS Dentate Gyrus Migratory Stream, DGML Dentate Gyrus Molecular Layer, HFA Hippocampus Fimbrial Angle, ps pial surface, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Migration, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 + cells in DGMS are TBR2 + neural progenitors. A High power picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 in the Hippocampal Fimbrial Angle (HFA). Double immunopositive, ACE2 and TBR2, cells are mainly localized in the basal surface of the VE (arrows) and in cells that are migrating into the subventricular zone (arrowhead). B Microphotograph showing the Dentate Gyrus Migratory Stream (DGMS) from the HFA to the pial surface (ps), where DGMS migratory cells follow dorsally to the Dentate Gyrus Hilux (DGH) or ventrally to Dentate Gyrus Molecular Layer (DGML). Insets localized the areas showed in ( A , C , and D ). C High power microphotograph showing ACE2 (DAB precipitate in the cytoplasm; arrowheads) and TBR2 (DAB-nickel precipitate in the nucleus; arrows) expression in ventricular and migratory cells. D High power of the zone represented in the inset of B showing double labeled ACE2 and TBR immunopositive cells in the DGMS (arrows). E Picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 at the Hippocampal Fimbrial Angle (HFA) (small arrows) and in migrating cells in the SVZ and DGMS (arrowheads). Large arrows show ACE2 and TBR immunopositive cells migrating near to a blood vessel (bv). F Low power picture of an immunofluorescence processed section, showing the DGMS at the caudal pole of the hippocampus, where the DG turns dorsally to became the fasciola cinerea. Ventral and dorsal DGMS are delimited by arrows. G , H High power picture of immunofluorescence showing ACE2 immunolabeling in the cytoplasm (green) and TBR2 immunolabeling in the nucleus (reed) in DGMS cells. Scale bar: A , C , E , G 10 µm: B 200 µm; D , H 7 µm; F 300 µm. bv blood vessel, CA Ammon’s horn, DGH Dentate Gyrus Hilux, DGML Dentate Gyrus Molecular Layer, DGMS Dentate Gyrus Migratory Stream, HFA Hippocampus Fimbrial Angle, ps pial surface, SVZ sub-ventricular Zone, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Labeling, Immunofluorescence, Immunolabeling

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 and neuropilin 1 (NP1) co-localize in the membrane of DGMS cells. A ACE2 immunofluorescent cells in the DGMS between sub-ventricular zone of the HFA and the ps (arrows). B High power confocal photomicrograph sowing the co-expression of ACE2 and NP1 in the membrane of migrating cells (arrows). The arrowhead shows the point where Z-stack projection have been reconstructed (view in the x and z plane), to demonstrate co-localization of ACE2 (reed dots) and NP1 (green/yellow dots) in the cellular membrane. C – E Microphotographs showing ACE2 and NP1 co-expression in DGMS cells. Inserts show the same cells with clear co-expression of ACE2 and NP1 in the membrane of leading processes (arrows). F – H DGMS cell showing strong co-expression of ACE2 and NP1 in the cellular membrane of leading processes (arrows). Scale bar: A : 250 µm; B 10 µ; C – E 20 µm; F – H 15 µm

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Membrane, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in migrating neural crest progenitors. A , B Anti-ACE2 immunofluorescence showed expression of ACE2 in the cytoplasm of cultured dental derived NCP, with perinuclear accumulation. A’ Dental derived NCP express Nestin protein (red immunofluorescence). B’ Cells with polarized morphology ACE2 expression is stronger and accumulated in the cellular membrane of the progression pole (arrows). C – I Scratch-wound assay in confluent cultures. D 10 min after the scratch (t0h) cells at the wound edge strongly expressed ACE2 (arrows). E – I At 12–24 h (t12h, t24h) after the scratch, polarized elongated cells migrated into the wound with strong expression of ACE2 in the cellular membrane of advance processes (arrows) including in intercellular nanotubes (arrows head in H ). I Quantification of ACE2 expression by immunofluorescence intensity and intracellular distribution with the ImageJ tool for measuring corrected total cell fluorescence (CTCF). Data shown as mean ± S.D. The number of cells analyzed were: T0h (cytoplasm or membrane) n = 26, T12h (cytosol or membrane) n = 22. Comparisons between cytoplasm and cell membrane at T0h and at T12h were made with tests for paired samples (Wilcoxon signed-rank test) and comparisons between cytoplasm at T0h and T12h or cell membrane at T0h and T12h were made with test for independent samples (Mann–Whitney test for T0h test), using the software GraphPad Prism. a.u arbitrary units. Asterisks indicate p -value: **** p < 0.0001 ns not significant. J At 48 h after the scratch the wound was completely cellularized with increased expression of ACE2 in some cell membranes of neighboring cells at the place where the wound was performed (arrows)

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Immunofluorescence, Cell Culture, Derivative Assay, Membrane, Scratch Wound Assay Assay, Fluorescence, MANN-WHITNEY, Software

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: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: High-throughput of ReFRAME, Pathogen Box, TargetMol and Cathepsin L drug libraries for SARS-CoV-2 antiviral compounds. A. Schematic of the spike protein of SARS-CoV-2. RBD: receptor binding domain. B. Schematic of the High-throughput assay. Compounds were pre-spotted in 1536-well plates. Next, 2000 HEK293T-ACE2 cells were added to each well and pre-incubated with each compound for 1 h, followed by infection with MLV reporter luciferase virus pseudotyped with the SARS-CoV-2 Spike protein (SARS2-S) or VSV-G protein (VSV-G). Luciferase was measured 48 h later. C. Summary of the ReFRAME library results. Conc.: concentration. D. Distribution of Z-Score for primary screens of each library. Scatter plot of Z_Score for all samples tested from the ReFrame library ( N = 1; circle) and other libraries ( N = 3; Cathepsin L: square; Pathogen Box: cross; TargetMol: filled circle). Total of 16,320 samples. Positive controls: orange; Negative control: cyan; Hit compounds: red; non-hit compounds: black. E. Summary of the 3 other libraries results. F. ReFrame library screening against different targets: SARS2-S, 3CLpro and PLpro. Venn diagram analysis of comparison between hits from SARS2-S entry, 3CLpro and PLpro assay against ReFRAME library results. There are 419 compounds that are SARS2-entry specific potential inhibitors. G. Robustness in terms of Z’ score of each screen for each library.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: High Throughput Screening Assay, Binding Assay, Incubation, Infection, Luciferase, Virus, Concentration Assay, Negative Control, Library Screening, Comparison

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Summary of the selected Cathepsin L, Pathogen box and TargetMol compounds in this study. Activity of the selected compounds against the different MLV pseudotyped viruses in HEK293-ACE2 cells and their respective cytotoxicity. Values for SARS2-S, VSV-G and toxicity are mean ± SEM of 2–4 independent experiments. TI: therapeutic index. * n = 1.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Targets of the selected compounds and SARS-CoV-2 wild type infection. A. Description of the targets of the different hits from all the studied libraries. B. Antiviral activity of the 2 best hits in the SARS-CoV-2-induced CPE assay. Vero E6 cells treated with test compounds for two hours were infected with SARS-CoV-2 at an MOI of 0.05, then incubated for three days in the presence of compound. Cell viability (protection from virus-induced CPE) was measured with CellTiter-Glo. C and D. Antiviral effect was measured with a subset of Vero E6 cells expressing a low (C) and high (D) level of ACE2. E. Cytotoxicity of selected compounds in Vero E6 cells. Cytotoxicity was tested in the same conditions with cell culture media instead of the virus. F. Virus yield reduction activity of selected compounds. Vero E6 cells infected with SARS-CoV-2 at an MOI of 0.05 were cultured in the presence of test compound (5 µM) and the supernatant was harvested after 24 and 48 h of incubation. The Progeny virus was enumerated with a plaque assay using an Avicel overlay in fresh Vero E6 cells. N = 3 experiments were performed for infectivity assays and n = 2 for the cytotoxicity assays. **** P < 0.0001, Two-way ANOVA with Dunnett's multiple comparisons test against DMSO control.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Infection, Activity Assay, Incubation, Virus, Expressing, Cell Culture, Plaque Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: SR-914 “calpeptin” specifically blocks SARS-CoV entry. A. Its activity against SARS2-S in HEK293T-ACE2-TMPRSS2 cells. Cells were incubated with different concentrations of drugs, then infected with SARS2-S or VSV-G. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 to 4 independent experiments. B. Time of drug addition experiment schematic. Infection was performed for 1 h with or without drugs, Vero CCL81 cells were then washed, and fresh media was added with or without drugs. C. Time of drug addition experiment result. SR-914 was used at 10 µM. E64d at 20 µM. Calp.: calpeptin = SR-914. NI: not infected. Shown is the mean ± SEM of 4 to 6 independent experiments. D. Luciferase complementation assay schematic. The reporter consists of a split Firefly luciferase protein connected by a cleavable peptide for the tested protease. Upon cleavage of the peptide, the luciferase protein undergoes dimerization for an active state. DnaE intein helps in this dimerization. E. Its activity against SARS2-S Entry, 3CLpro and PLpro. C-: negative control. C+; positive control. Shown is the mean ± SD of 3 independent experiments. One-way ANOVA followed by Tukey's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Negative Control, Positive Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Breath of activity of calpeptin against various SARS-CoVs. A. Its activity against SARS1-S in HEK293T-ACE2 cells. HEK293T-ACE2 cells were incubated with different concentrations of calpeptin, then infected with SARS1-S. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 independent experiments. B. Schematic of the substituted residues in the S protein of the highest threat of SARS-CoV-2 strains. C. Evolution of the S protein residues at the position 417, 484, 501 and 614 from 2019 to February 2021. Modified figure from https://nextstrain. org/ncov/global?branchLabel=none& c =gt-S_417,484,501,614& l =clock. D. Activity of the new emergent variants. HEK293T-ACE2 cells were infected with different mutants of SARS2-S. The day after, a medium change was performed. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 3 independent experiments. WT: wild type, SA: South Africa, UK: United Kingdom. E. Activity of calpeptin activity against crucial mutations present in the S protein of the new emergent strains. Similar experiment than D but calpeptin was added during infection and after medium change. Shown is the mean ± SEM of n = 2–5 independent experiments. Two-way ANOVA followed by Dunnett's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Modification

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Quantification of VSV(luc)ΔG*SARS-CoV-2-S entry by measuring luciferase activity in HEK293T cells transiently expressing the indicated IFITM proteins. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in supplementary data . b Calu-3 cells treated with non-targeting (CTRL) or IFITM1, 2, or 3 siRNAs or a combination of the three and infected with VSV(luc)ΔG*SARS-CoV-2-S particles. Bars represent the mean of three independent experiments (±SEM). c Quantification of viral N RNA levels by qRT-PCR 48 h post-infection with SARS-CoV-2 (MOI 0.05) in the supernatant of HEK293T cells transiently expressing ACE2 alone or together with the indicated IFITM proteins. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in the supplementary data . d Quantification of viral N RNA levels by qRT-PCR in the supernatant of Calu-3 cells, collected 48 h post-infection with SARS-CoV-2 (MOI 0.05). Cells were transfected with control (CTRL) or IFITM1, 2, and/or 3 targeting siRNA or a combination of the three and either treated with IFN-β or left untreated as indicated. Bars represent the mean of four independent experiments (±SEM). *** p < 0.0001. e Infectious SARS-CoV-2 particles in the supernatant of ( d ) as assessed by TCID50 assay. Bars represent the mean of three independent experiments (±SEM), exact p values are provided in Supplementary Data . f Infectious SARS-CoV-2 particles in the supernatant of ( d ) as assessed by plaque-forming unit assay. g Quantification of ( f ). Bars represent the mean of three independent experiments (±SEM). siRNA CTRL vs. siRNA IFITM1 p = **0.0012, siRNA CTRL vs. siRNA IFITM2 p = ****<0.0001, siRNA CTRL vs. siRNA IFITM3 p = *0.0197, siRNA CTRL vs. siRNA IFITM1–3 p = **0.0020). h , i Correlation of infectious viral particle analysis ( h , TCID50; i , plaques) of ( e , g ) with qPCR data from ( d ) values showed calculated as a Pearson correlation ( h : r = 0.82, p < 0.0001, i : r = 0.83, p = 0.0002). a , c , d , g Unpaired t test with Welch’s correction. b , e Unpaired t tests. p Values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Calu-3 cells were seeded in 48-well format (peptides assays), or in 24-well format (antibodies assay), 24 h later cells were treated with increasing concentrations (20 and 80 µg/ml) of IFITMs derived peptides (human IFITM2 long: EEQEVAMLGVPHNPAPPMSTVIH, human IFITM2 short: QEVAMLGVPHNAPPMST-VIH, mouse IFITM2 (mIFITM2) long: EEYGVTELGEPSNSAVVRTTVIN, human IFITM3 long: EEHEVAVLGAPHNPAPPTSTVIH, scrambled IFITM2: EGESGVTTATVEVVIER-NN-LPY) or blocking antibodies (15 and 30 µg/ml) (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668; α-IFITM1 Cell Signaling 13126 S, α-IFITM2 Cell Signaling 13530 S, α-IFITM3 Proteintech 11714-1-AP, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026) as indicated.

Techniques: Luciferase, Activity Assay, Expressing, Infection, Quantitative RT-PCR, Transfection, TCID50 Assay

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Quantification and exemplary images of a PLA between SARS-CoV-2 Spike and ACE2 in Calu-3 depleted of IFITM1, IFITM2, or IFITM3 and infected with genuine SARS-CoV-2. Bars represent the mean of four independent experiments (100 cells ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. b Quantification and exemplary images of a PLA between Spike and ACE2 in Calu-3 cells depleted of IFITM2 and infected with SARS-CoV-2 virus on ice for 2 h and then incubated for 15 min at 37 °C. Bars represent the mean of four independent experiments (300 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. c Quantification and exemplary images of a PLA assay between Spike and RAB5A as in ( c ). Bars represent the mean of four independent experiments (400 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. DAPI (blue), nuclei. PLA signal (yellow). Scale bar, 20 µm. d Summary of the quantification shown in panels ( b ) (Spike-ACE2) and ( c ) (Spike-RAB5α) proximity upon SARS-CoV-2 infection. Individual data points and statistics are provided in panels ( b ) and ( c ). e Exemplary images of the colocalization (white) of PLA foci (red) indicating SARS-CoV-2 Spike and IFITM2 with early (EEA1, green, upper panel) or late endosomal markers (Rab7a, green, lower panel) in Calu-3 cells. Cells were infected with SARS-CoV-2 for 2 h at 4 °C or 2 h at 4 °C and 15 min at 37 °C as indicated. DAPI (blue), nuclei. f Quantification of the percentage of colocalization between the PLA signal and the indicated endosomal markers in 225 × 225 µm images. Bars represent the mean of four individual images (±SEM), unpaired t test, ** p = 0.0031. g Quantification of the combined PLA (IFITM2/Spike) in ( e ). Bars represent the mean of 120 cells (±SEM) over two independent experiments. The experiment was replicated twice to similar results. Two-sided Wilcoxon matched-pairs test, **** p < 0.0001. a – c , e Scale bars, 20 μm.

Article Snippet: Calu-3 cells were seeded in 48-well format (peptides assays), or in 24-well format (antibodies assay), 24 h later cells were treated with increasing concentrations (20 and 80 µg/ml) of IFITMs derived peptides (human IFITM2 long: EEQEVAMLGVPHNPAPPMSTVIH, human IFITM2 short: QEVAMLGVPHNAPPMST-VIH, mouse IFITM2 (mIFITM2) long: EEYGVTELGEPSNSAVVRTTVIN, human IFITM3 long: EEHEVAVLGAPHNPAPPTSTVIH, scrambled IFITM2: EGESGVTTATVEVVIER-NN-LPY) or blocking antibodies (15 and 30 µg/ml) (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668; α-IFITM1 Cell Signaling 13126 S, α-IFITM2 Cell Signaling 13530 S, α-IFITM3 Proteintech 11714-1-AP, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026) as indicated.

Techniques: Infection, Incubation

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Alignment of the amino acid sequence of human IFITM1, 2, and 3. Binding sites of IFITM blocking antibodies are indicated and the region of origin of the IFITM derived peptides highlighted. b Quantification of viral N RNA levels in the supernatant of Calu-3 cells treated with α-ACE2, α-IFITM1, α-IFITM2, α-IFITM3, and α-IFITM1-3 antibodies (7.5, 15, or 30 µg/ml) 1 h before infection (SARS-CoV-2, MOI 0.05), collected 48 h post-infection. Bars represent the mean of two (30 µg/ml α-IFITM2) or three (all other concentrations) independent experiments each measured in technical duplicates (±SEM), unpaired t-test. All p values were calculated compared to CTRL (ACE2, 15 µg/ml: 0.0011, ACE2, 30 µg/ml: <0.00001; IFITM2, 30 µg/ml: <0.00001; IFITM1-3, 15 µg/ml: 0.004; IFITM1-3, 30 µg/ml: <0.00001). c Quantification of viral N RNA levels in the supernatant of Calu-3 cells treated with IFITM-derived peptides (20 or 80 µg/ml) as indicated for 1 h before infection (MOI 0.05), collected 48 h post-infection. Bars represent the mean of three (hIFITM2 long and short peptide) or four (CTRL, hIFITM3 and scrambled) independent experiments each measured in technical duplicates (±SEM), unpaired t test. All p values were calculated compared to CTRL (hIFITM2 long, 20 µg/ml: 0.005; hIFITM2 long, 80 µg/ml: <0.0001; hIFITM2 short, 20 µg/ml: 0.0009; hIFITM2 short, 80 µg/ml: 0.0002; hIFITM3, 20 µg/ml: 0.0031). p Values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 or were not significant ( p > 0.05).

Article Snippet: Calu-3 cells were seeded in 48-well format (peptides assays), or in 24-well format (antibodies assay), 24 h later cells were treated with increasing concentrations (20 and 80 µg/ml) of IFITMs derived peptides (human IFITM2 long: EEQEVAMLGVPHNPAPPMSTVIH, human IFITM2 short: QEVAMLGVPHNAPPMST-VIH, mouse IFITM2 (mIFITM2) long: EEYGVTELGEPSNSAVVRTTVIN, human IFITM3 long: EEHEVAVLGAPHNPAPPTSTVIH, scrambled IFITM2: EGESGVTTATVEVVIER-NN-LPY) or blocking antibodies (15 and 30 µg/ml) (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668; α-IFITM1 Cell Signaling 13126 S, α-IFITM2 Cell Signaling 13530 S, α-IFITM3 Proteintech 11714-1-AP, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026) as indicated.

Techniques: Sequencing, Binding Assay, Blocking Assay, Derivative Assay, Infection

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Immunohistochemistry of gut organoids treated with blocking antibodies and a mouse IFITM2 (mIFITM2) derived peptide and infected with SARS-CoV-2 (MOI 0.15). Organoids were stained with α-SARS-CoV-2 N (red), E-Cadherin (green), and DAPI (blue). Scale bar, 100 µm (left panel). Quantification of SARS-CoV-2 N fluorescence, normalized to DAPI (right panel). Bars represent the mean of CTRL:9, α-ACE2 15 µg/ml:8, α-ACE2 30 µg/ml:2, α-IFITM1-3 15 µg/ml:5, α-IFITM1-3 30 µg/ml:6, mIFITM2 peptide 15 µg/ml:6, or mIFITM2 peptide 30 µg/ml:2 individual images over one experiment (±SEM), unpaired t-test with Welch’s correction All p values were calculated compared to CTRL (α-ACE2 15 µg/ml: 0.0104, α-ACE2 30 µg/ml: 0.0093, α-IFITM1-3 30 µg/ml: 0.0002, mIFITM2 peptide 15 µg/ml: 0.0295, mIFITM2 peptide 30 µg/ml: 0.005). b Quantification of viral N RNA levels in the supernatant of SARS-CoV-2 infected cardiomyocytes (increasing MOIs as indicated) at indicated time points. Lines represent a single experiment measured in duplicates. c Immunoblot of IFITM1, IFITM2, and IFITM3 in cardiomyocytes infected with SARS-CoV-2. Whole-cell lysates were stained with α-IFITM1, α-IFITM2, α-IFITM3, and α-GAPDH. n = 1. d Quantification of viral N RNA levels in the supernatant of SARS-CoV-2 infected cardiomyocytes (MOI 0.1) treated with IFITM-derived peptides, collected at indicated time points post-infection. Bars represent the mean of two independent experiments each measured in technical duplicates. Non-infected controls were below the quantification level (<1000). Exact p values are provided in Supplementary Data . p Values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 or were not significant ( p > 0.05).

Article Snippet: Calu-3 cells were seeded in 48-well format (peptides assays), or in 24-well format (antibodies assay), 24 h later cells were treated with increasing concentrations (20 and 80 µg/ml) of IFITMs derived peptides (human IFITM2 long: EEQEVAMLGVPHNPAPPMSTVIH, human IFITM2 short: QEVAMLGVPHNAPPMST-VIH, mouse IFITM2 (mIFITM2) long: EEYGVTELGEPSNSAVVRTTVIN, human IFITM3 long: EEHEVAVLGAPHNPAPPTSTVIH, scrambled IFITM2: EGESGVTTATVEVVIER-NN-LPY) or blocking antibodies (15 and 30 µg/ml) (α-ACE2 AK (AC18Z), Santa Cruz Biotechnology sc-73668; α-IFITM1 Cell Signaling 13126 S, α-IFITM2 Cell Signaling 13530 S, α-IFITM3 Proteintech 11714-1-AP, α-IFITM1/2/3 (F-12) Santa Cruz Biotechnology sc-374026) as indicated.

Techniques: Immunohistochemistry, Blocking Assay, Derivative Assay, Infection, Staining, Fluorescence, Western Blot