Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Time-dependent global cellular sphingolipid (SL) changes upon infection with three different CoVs. ( A ) Experimental design of the sphingolipidome analysis. Huh-7-ACE2 cells were mock infected or infected with the indicated CoV (multiplicity of infection [MOI] = 3) for 1, 6, and 12 hpi. ( B and C ) Corresponding growth kinetics and immunofluorescence images. Scale bars = 100 µm. ( D ) Heat maps showing fold changes of deregulated SL species at the indicated time points in relation to uninfected control (significant differences [ P ≤ 0.05] in bold and marked with asterisks) calculated from the replicates by one-way analysis of variance (ANOVA) with Dunnett´s test for multiple comparisons. ( E ) Corresponding Venn diagrams. Experiments were done in quintuplicates ( n = 5). ( F ) Simplified illustration of SL metabolism. Ceramide (Cer), as the centerpiece of the SL metabolic pathway, can be synthesized de novo via dhCer, via salvage pathway through hydrolysis of glycosphingolipids or by the sphingomyelinase (SMases) pathway through the hydrolysis of SM. Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SM, sphingomyelin; Sph, sphingosine.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Immunofluorescence, Control, Synthesized

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Antiviral activities of a/nSMase inhibition in CoVs replication. ( A through D ) Huh-7-ACE2 cells were mock-infected ( A and C ) or infected with the indicated virus (MOI = 0.1; B and D ) in the presence of increasing concentrations of SMase inhibitors ([ A and B ] ARC39 for aSMase and [ C and D ] PDDC for nSMase2) or dimethyl sulfoxide (DMSO) as solvent control. Cell viability ( A and C ) or virus titers ( B and D ) in the presence of increasing inhibitor concentrations were determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay or plaque assay. ( E and F ) Genetic manipulation of SMases or CoV-specific entry receptors using siRNA knockdown. ( E ) Huh-7-ACE2 cells were transfected with the indicated siRNAs, and the target mRNA was analyzed using qPCR. ( F ) Impact of siRNA silencing on viral replication. Huh-7-ACE2 cells were reverse transfected with siRNAs (100 nM) for 48 h before being infected with the indicated virus. Infectivity was assessed by image-based quantification of N-positive cells and was normalized to levels in cells targeted by scrambled (scr) siRNA controls. All experiments were performed in Huh-7-ACE2 cells mock-infected or infected with the indicated virus at an MOI of 0.1 in three independent replicates. All bar graphs show mean ± SD; asterisks indicate P values (* P < 0.05; ** P < 0.005; *** P < 0.0005) obtained by a two-tailed unpaired t -test.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Inhibition, Infection, Virus, Solvent, Control, Plaque Assay, Knockdown, Transfection, Two Tailed Test

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Time-dependent antiviral effects of nSMase2 inhibitor on coronaviral RO formation. ( A ) HCoV-229E-infected Huh-7-ACE2 cells (MOI = 3) were treated with PDDC (10 µM) for different time periods post-infection as indicated below. Production of infectious virus progeny was determined using (pooled) cell culture supernatants collected until 12 hpi. Virus titers were determined and compared to the titer determined for infected but untreated cells. ( B through E ) Huh-7-ACE2 cells were infected with HCoV-229E and then either left untreated ( C ), or treated with PDDC (10 µM, D ) or K22 (40 µM, E ) for 8 hpi. Subcellular replication sites were identified by a double-stranded RNA (dsRNA)-specific antibody in the presence or absence of the indicated inhibitor. Nuclei were stained using DAPI. ( B ) Quantification of RO-positive cells by image-based quantification of dsRNA-positive cells in relation to total cell count. All bar graphs show mean ± SD; asterisks indicate P values (n.s., not significant; * P < 0.05; ** P < 0.005; *** P < 0.0005) obtained by a two-tailed unpaired t -test. ( C through E ) Corresponding representative images from one out of three independent experiments. The scale bar in the second row represents 5 µm. All experiments were performed in three independent replicates.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Virus, Cell Culture, Staining, Cell Counting, Two Tailed Test

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of ROs and sphingolipids in CoV-infected cells. ( A ) Huh-7-ACE2 cells were transfected with an Eqt-SM-oxGFP expression construct (to visualize SM, green) and after 48 h infected with the indicated CoV (MOI = 3). Eight hours post-infection, cells were fixed and stained for viral ROs (red) using an antibody against dsRNA, a specific marker for viral replication intermediates. ( B ) Huh-7-ACE2 cells were infected with the indicated CoV (MOI = 3) for 8 hpi, fixed, and permeabilized using 0.5% saponin. Cells were stained for viral ROs (dsRNA, red) using an antibody against dsRNA and an antibody against Cer (green). DAPI was used for staining of nuclei. Insets indicate regions of interest displayed at higher magnification in the next row. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Transfection, Expressing, Construct, Staining, Marker

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of CoV-induced ROs with nSMase2. ( A ) Huh-7-ACE2 cells were transfected with an nSMase2-eGFP-expressing construct (to visualize sphingomyelinase, green) and infected with HCoV-229E, SARS-CoV-2, or MERS-CoV (MOI = 3) and fixed 8 hpi with 3.7% paraformaldehyde (PFA). Viral ROs (red) were stained using an antibody against dsRNA. ( B ) Huh-7-ACE2 cells were infected with HCoV-229E (MOI = 3) and treated as indicated with the nSMase2 inhibitor PDDC. Viral ROs (red) and ceramide (green) were stained using respective antibodies. Filled arrows indicate colocalization. Outline arrows indicate ceramide spots without a dsRNA signal. DAPI was used for staining of nuclei. Insets indicate regions of interest displayed at higher magnification in the next row. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Expressing, Construct, Infection, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Artificially induced ROs by overexpressing a self-cleaving HCoV-229E nsp3-4 construct. ( A ) Schematic illustration of constructs generated to induce artificial HCoV-229E ROs upon transfection. The epitope tags used at the termini of the constructs are indicated as dots. The HA-nsp3-4-V5_K2481A construct contains an alanine substitution in the cleavage site of nsp3-4, therefore avoiding nsp3-mediated polyprotein cleavage. The HA-nsp3-4-V5_C1701A construct contains an alanine substitution that abrogates PL pro activity. ( B ) HEK-293T-ACE2 cells were transfected for 24 h with the indicated expression constructs, lysed, and HA-nsp3 and nsp4-V5-tagged proteins were detected using Western blot analysis. GAPDH served as a loading control. ( C ) Huh-7-ACE2 cells were transfected for 24 h with the indicated expression constructs, fixed, and stained using HA-specific (green) or V5-specific (red) antibodies. Subcellular localization was visualized by confocal microscopy using a Leica SP05. DAPI was used for staining of nuclei. Scale bars = 5 µm. ( D ) Huh-7-ACE2 cells were transfected with the indicated constructs, fixed 24 hours post-transfection, and analyzed via transmission electron microscopy analysis using a Zeiss LEO electron microscope. Scale bars = 500 nm.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Construct, Generated, Transfection, Activity Assay, Expressing, Western Blot, Control, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Microscopy

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of artificially induced ROs and Cer. ( A and B ) Huh-7-ACE2 cells were transfected with the indicated plasmids (0.75 µg DNA) expressing either HA-nsp3-4-V5 or mutants (HA-nsp3-4-V5_K2481A and HA-nsp3-4-V5_C1701A) ( A ) or the single constructs ( B ). After 24 h, the cells were fixed with 3.7% paraformaldehyde (PFA). The cells were then permeabilized with 0.5% saponin. Nsp3 or 4 (red) and Cer (green) were visualized using HA- (nsp3), V5- (nsp4), and Cer- specific antibodies. ( C and D ) Huh-7-ACE2 cells were transfected with the indicated plasmids (0.75 µg DNA) expressing nSMase_eGFP (green) and either HA-nsp3-4-V5 or mutants (HA-nsp3-4-V5_K2481A and HA-nsp3-4-V5_C1701A; C ) or the single constructs ( D ). After 24 h, the cells were fixed with 3.7% PFA. The cells were then permeabilized with 0.5% saponin. Nsp3 or 4 (red) visualized using HA- (nsp3) or V5- (nsp4) specific antibodies. DAPI was used for staining of nuclei. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Representative images from one out of three biologically independent experiments were shown. Scale bars = 5 µm.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Expressing, Construct, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Overview of sphingolipid (SL) changes upon artificial RO formation upon transfection with constructs expressing nsp3 and nsp4 in Huh-7-ACE2 cells. (A) Experimental design of the sphingolipidome analysis. Huh-7-ACE2 cells were mock transfected with empty vector control (pcDNA3.1) or transfected with either HA-nsp3-4-V5, HA-nsp3-4-V5_K2481A or HA-nsp3-4-V5_C1701A for 24 h. (B) Corresponding immunofluorescence images of transfected cells. The value indicates transfection efficacy of HA-nsp3-positive cells (green) in relation to total cell count. Scale bars = 100 µm. (C) Heatmap showing fold changes of deregulated SL species in relation to mock-transfected control (significant differences in bold and marked with asterisks, P ≤ 0.05). Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SL, sphingolipid; SM, sphingomyelin; Sph, sphingosine

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Control, Immunofluorescence, Cell Counting

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of CoV-induced ROs and Cer in lung-derived cells. (A) Adenocarcinoma cell line A549-ACE2 (for SARS-CoV-2) or A549-CD13 (for HCoV-229E) or (B) primary lung fibroblasts MRC-5 cells (for HCoV-229E and MERS-CoV) were infected with an MOI of 3 for 8 hpi. The fixed cells were then permeabilized with 0.5% saponin and stained against dsRNA (red) and Cer (green). DAPI was used for staining of nuclei. Colocalization signals, rates and MCCs were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Derivative Assay, Infection, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Overview of sphingolipid changes upon infection with HCoV-229E and SARS-CoV-2 in lung-derived cells. (A) Experimental design of the sphingolipidome analysis. A549-ACE2 or A549-CD13 cells were mock-infected or infected with HCoV-229E (A549-CD13) or SARS-CoV-2 (A549-ACE2) with an MOI of 3 for 12 hpi. (B and C) Corresponding viral titers and immunofluorescence images of A549-ACE2 (for SARS-CoV-2) and A549-CD13 (for HCoV-229E) cells (MOI = 3) 12 hpi. Scale bars = 100 µm. (D) Heatmap showing fold changes of deregulated sphingolipid species at the indicated time points in relation to uninfected control based on significant differences (significant differences in bold and marked with asterisks, P ≤ 0.05) calculated from the replicates by t -test (SARS-CoV-2) or one-way ANOVA with Dunnett´s test for multiple comparisons (HCoV-229E). (E) Corresponding Venn diagrams. Experiments were done in biological independent replicates ( n = 5). Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SL, sphingolipid; SM, sphingomyelin; Sph, sphingosine.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Derivative Assay, Immunofluorescence, Control

Journal: Cell Reports

Article Title: SARS-CoV-2 reactive and neutralizing antibodies discovered by single-cell sequencing of plasma cells and mammalian display

doi: 10.1016/j.celrep.2021.110242

Figure Lengend Snippet: Characterization of RBD-specific antibodies for affinity, epitope binning, and neutralization (A–C) Purified antibodies were assayed at different concentrations (shown here: 125, 37.5, 7.8, and 1.9 nM). Software-calculated fits are shown in red. Binding kinetics (apparent affinity constant k D ) to SARS-CoV-2 S1 for FACS-confirmed RBD binders, determined by biolayer interferometry. (D) Epitope binning setup of binders with ACE2. (E) Additional binding by the secondary molecule indicates an unoccupied epitope (non-competitor, green), while no binding indicates epitope blocking (competitor, red). Self-blocking confirmation can be found on the diagonal. (F) Viral neutralization, reduction in luciferase signal indicates successful viral neutralization. (G) Relative response of RBD binders to variants Alpha, Beta, Gamma, Delta, Delta+, Epsilon, and Kappa.

Article Snippet: Cells were subcultured every 48-72 h. ACE2 + Human Embryonic Kidney cells (HEK293T-ACE2, derived from ATCC CRL-3216) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Cat#11960-085) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin at 37°C 5% CO 2 .

Techniques: Neutralization, Purification, Software, Binding Assay, Blocking Assay, Luciferase

Journal: Cell Reports

Article Title: SARS-CoV-2 reactive and neutralizing antibodies discovered by single-cell sequencing of plasma cells and mammalian display

doi: 10.1016/j.celrep.2021.110242

Figure Lengend Snippet:

Article Snippet: Cells were subcultured every 48-72 h. ACE2 + Human Embryonic Kidney cells (HEK293T-ACE2, derived from ATCC CRL-3216) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Cat#11960-085) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin at 37°C 5% CO 2 .

Techniques: Virus, Recombinant, Multiplex Assay, Selection, Sequencing, Construct, Expressing, Software

Journal: bioRxiv

Article Title: Therapeutic efficacy of CT-P59 against P.1 variant of SARS-CoV-2

doi: 10.1101/2021.07.08.451696

Figure Lengend Snippet: Serially diluted CT-P59 was mixed with indicated variant pseudoviruses for inoculation to ACE2-expressing HEK293T cells. Luciferase activity was measured and %Neutralization was calculated (A). CT-P59 dilutions were pre-incubated with P.1 variant. The antibody-virus mixture were inoculated into Vero E6 cells, incubated and probed by anti-nucleocapsid antibody and staining (B). VC and CC represent as virus control and cell control, respectively.

Article Snippet: Derivatives of HEK-293T cells expressing ACE2 were generated by transducing HEK-293T (ATCC, CRL-3216) cells with ACE2 (Addgene, #145839).

Techniques: Variant Assay, Expressing, Luciferase, Activity Assay, Neutralization, Incubation, Virus, Staining, Control

Journal: bioRxiv

Article Title: Therapeutic efficacy of CT-P59 against P.1 variant of SARS-CoV-2

doi: 10.1101/2021.07.08.451696

Figure Lengend Snippet: Human ACE2 Transgenic mice (n=11/group) were challenged with 10 4 PFU of P.1 variants. Vehicle and 5, 20, 40 and 80 mg/kg of CT-P59 were administered intraperitoneally 8 h after virus inoculation. Body weights and survival rates were monitored daily until 10 dpi (A, B). 30% or higher of weight loss was considered to be dead. Four animals each were euthanized for virus titration at 3 dpi and 6 dpi. The virus titers from lung tissue and nasal wash were measured (C, D, E, F) using plaque assay. Alphabets and asterisks indicates statistical significance between the control and each groups as determined by one-way ANOVA followed by a Dunnett’s post-hos test. a denotes p<0.0001 between control and CT-P59 treatment groups (pooled). b denotes p<0.01 to p<0.05 between control and CT-P59 treatment group at 80 mg/kg. c denotes p<0.0001 to p<0.01 between control and CT-P59 treatment groups at 5, 20, 40 and 80 mg/kg. d denotes p<0.0001 to p<0.001 between control and CT-P59 treatment groups at 5, 20, 40 and 80 mg/kg. * indicates P < 0.05, and **** indicates P < 0.0001.

Article Snippet: Derivatives of HEK-293T cells expressing ACE2 were generated by transducing HEK-293T (ATCC, CRL-3216) cells with ACE2 (Addgene, #145839).

Techniques: Transgenic Assay, Virus, Titration, Plaque Assay, Control