Journal: Frontiers in Cardiovascular Medicine

Article Title: SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity

doi: 10.3389/fcvm.2021.687783

Figure Lengend Snippet: Spike increases Rab5a association with ACE2 and junctional proteins. (A) Representative Western blot of coimmunoprecipitation experiments done using Rab5a antibody and probed for different proteins. (B) Mean data indicating fold change in Rab5a association with junctional proteins and ACE2 after S1RBD treatment. n = 6 for each, * P < 0.05 vs. untreated control. (C) Representative Western blot of coimmunoprecipitation experiments done using ACE2 antibody and probed for Rab5a. (D) Mean data indicating fold change in ACE2 association with Rab5a after Spike treatment. n = 6 for each, * P < 0.05 vs. untreated control. (E) Representative Western blot of coimmunoprecipitation experiments in control and diabetic cells after Rab5a knockdown with siRNA. (F) Mean data indicating fold change in ACE2 association with Rab5a after Rab5a siRNA. n = 6 for each, * P < 0.05 vs. control, # P < 0.05 vs. NT siRNA diabetic.

Article Snippet: Membranes were blocked with 5% non-fat milk and incubated with the one of the following primary antibodies: VE-Cadherin, PECAM1 (Novus Biologics), JAM-A (Invitrogen), Connexin-43, Rab5a (Cell Signaling Inc.), ACE2 (Abnova), or Actin (Millipore Sigma) overnight at 4°C.

Techniques: Western Blot, Control, Knockdown

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet: Stringent ACE2 requirement for pancreatic islet cell infection with SARS-CoV-2 (A) Representative contour plots gated on live α, β, and “other” cells pre-treated with IgG (irrelevant polyclonal goat antibody AF7197) or the anti-ACE2 blocking antibody AF933 prior to SARS-CoV-2 infection (48 h). (B) Summary of SARS-CoV-2 NP expression by live islet cell subsets as a function of IgG treatment or ACE2 blockade (n = 6 donors). (C) Percent infection inhibition for β and “other” cells (inhibition for α cells is not shown because the very low extent of α cell infection in IgG-treated cultures for 2 of 6 donors substantially skews such calculations). (D) Infectious SARS-CoV-2 titers and extent of infection inhibition following ACE2 blockade (n = 3 donors). (E) Quantification of chemokines and cytokines in UV-inactivated TCS of SARS-CoV-2-infected islet cell cultures under conditions of IgG treatment or ACE2 blockade (48-h infection, n = 3 donors). (F) Infectious SARS-CoV-2 titers in TCS as a function of glucose concentration in islet culture medium (n = 3 donors). (G) Quantification of CXCL10 and CXCL11 in TCS as a function of glucose concentration. All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable.

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Infection, Blocking Assay, Expressing, Inhibition, Concentration Assay

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

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, 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: 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: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, Cells transfected with ACE2, L-SIGN, DC-SIGN, and CD147 (red line) or mock (shaded gray) were stained with SCoV2-NTD-Fc and RBD-Fc fusion protein (10 μg/mL). Each transfectant was also stained with a specific monoclonal antibody. b, Effect of mannan and anti-CD209 (anti-DC-SIGN) antibody on SCoV2-NTD-Fc binding to DC- and L-SIGN transfectants or mock (shaded gray). The transfectants were preincubated with mannan (light blue line) or anti-CD209 antibody (dark blue), followed by the staining with NTD-Fc fusion protein. c, Staining of DC- and L-SIGN transfectants (red line) or mock (shaded gray) with SCoV2-NTD-Fc and SCoV-NTD-Fc fusion proteins (10 μg/mL). d, Cells transfected with flag-tagged spike proteins of SCoV2, SCoV, human coronavirus OC43, or HKU1 (red line) or mock (shaded gray) were stained with DC-, L-SIGN-Fc fusion proteins or anti-Flag-tag antibody. Proportions of the stained cells are shown. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Transfection, Staining, Binding Assay, FLAG-tag

Journal: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, SCoV2-PV infection on DC-SIGN, L-SIGN, mock, or ACE2 transfectants and Vero E6 cells. SCoV2-PV carrying a luciferase gene was used for the infection and luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.0018; ** P = 0.0003 b, DC-SIGN or L-SIGN transfectants were infected with recombinant SCoV2 with the NanoBiT luciferase gene. The luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.001; ** P = 0.0006. c, Cell-cell fusion assay of SCoV2 spike transfectants and DC- or L-SIGN transfectants. The effector cells expressing spike and T7 polymerase were cocultured with target cells expressing DC-SIGN, L-SIGN, mock, and T7 promoter-driven luciferase. Luciferase activities were measured after 24 h. Asterisks indicate statistical significance derived from unpaired T-test *** P <0.0001. d, Cell-cell fusion assay of SCoV2 spike transfectants (red) and DC-SIGN transfectants (green). Representative images are shown. Scale bar represents 50 μm in length. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Infection, Luciferase, Activity Assay, Derivative Assay, Recombinant, Cell-Cell Fusion Assay, Expressing