Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 1 | SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Binding Assay, Clone Assay, Marker, Magnetic Beads

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 5 | SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B), except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase If, a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Western Blot, Activity Assay, Magnetic Beads, Labeling, Positive Control

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 6 | Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacIq (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot

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: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

doi: 10.1038/s41467-021-22781-1

Figure Lengend Snippet: a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

Article Snippet: For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

Techniques: Expressing, Immunohistochemistry

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Antibodies used for immunofluorescence staining.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence, Staining

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Composition of antibodies used in the multiplexed immunofluorescence panels.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: ( A , B ) Multiplex imaging analysis depicting expression of ACE2, the major receptor for SARS-CoV-2, in different cellular compartments of lung tissues in a representative patient with COVID-19 (case #6). ( A ) Red arrows point to ACE2-expressing pneumocytes, defined by double-expression of ACE2 (yellow) and PANCK (white). ( B ) Cyan arrows highlight ACE2-expressing macrophages, defined by double-expression of ACE2 (yellow) and CD68 (red). All images were acquired on the Vectra Polaris microscope using a 20x objective magnification, and zoomed in areas are depicted for visualization of macrophages. ( C ) The was no difference in the relative frequencies (% of total imaged cells) of CD3+ T cells in lung control tissues (non-COVID-19 DAD, n = 5) and COVID-19 lung tissues ( n = 5), expressed as mean values with SD at the scatter dot plots (Mann–Whitney U test, p = 0.87). ( D ) There was no statistically significant difference of the CD4+ to CD8+ T lymphocyte ratio between the two cohorts (Mann–Whitney U test, p = 0.55).

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Multiplex Assay, Imaging, Expressing, Microscopy, MANN-WHITNEY

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