human ace2 receptor  (ATCC)

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: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: ODE single and repetitive exposure increases murine lung soluble ACE levels dependent upon ADAM-17 with associated effect on SARS-CoV-2 pseudovirus infectivity. Scatter plots with bars depict mean with SEM of lung ACE2 levels of ( A ) wild-type (WT) mice following single and repetitive (13 times) ODE exposures and ( B ) humanized ACE2 mice treated with or without TAPI- 1, an ADAM-17 inhibitor, prior to single instillation with saline or 12.5% ODE exposure ( n = 6 mice/group). Humanized ACE2 mice were exposed to a single dose or repetitive doses of ODE prior to SARS-CoV-2 pseudovirus (PV) infection with lungs collected 5 days post-infection. ( C ) Scatter plot with mean and SEM depicted viral titer determined by qPCR ( n = 6–8 mice/group). * p < 0.05, ** p < 0.01, **** p < 0.0001; groups compared using Student’s t -test in ( A ) and one-way ANOVA with Tukey’s post hoc test in ( B , C ).

Article Snippet: Eight-week-old wild-type (WT) or transgenic mice expressing the human ACE2 receptor (Jackson Labs, Bar Harbor, ME USA) were used for all animal experiments.

Techniques: Infection, Saline

Journal: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: Inhibition of PKCα or ADAM-17 along with ODE treatment synergistically increases membrane ACE2 levels enhancing SARS-CoV-2 pseudovirus entry in BEAS-2B cells in vitro. ( A ) Wild-type (WT) and PKCα-deficient (DN) BEAS-2B cells were treated with Gö 6976 (a PKCα inhibitor), TAPI-1 (an ADAM-17 inhibitor), 0.5% ODE, or a combination for 1 h in vitro. The cells were then collected and stained for flow cytometry analysis of membrane ACE2 expression. ( B ) Treated cells were infected for 48 h with SARS-CoV-2 pseudovirus expressing fluorescent dTomato. The cells were then fixed, stained with Hoechst nuclear stain, and analyzed using Operetta CLS. WT cells treated with both ODE and inhibitor had significantly higher infection than single-treated WT groups. ( C ) Representative flow cytometry images showing ACE2 gating. ( D ) Representative immunofluorescence images from Operetta CLS showing pseudovirus-infected cells (20× magnification; scale bar: 100 μm). Data shown are mean ± SEM; n = 9 per group; experiments were repeated 4 times; ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA with Tukey’s post hoc test).

Article Snippet: Eight-week-old wild-type (WT) or transgenic mice expressing the human ACE2 receptor (Jackson Labs, Bar Harbor, ME USA) were used for all animal experiments.

Techniques: Inhibition, Membrane, In Vitro, Staining, Flow Cytometry, Expressing, Infection, Immunofluorescence

Journal: International journal of translational medicine (Basel, Switzerland)

Article Title: Organic Dust Exposure Enhances SARS-CoV-2 Entry in a PKC α - and ADAM-17-Dependent Manner

doi: 10.3390/ijtm4030032

Figure Lengend Snippet: Proposed mechanism through which agricultural dust exposure could affect SARS-CoV-2 entry in vitro (created with BioRender.com ). Organic dust exposure (ODE) activates Toll-like receptor 2 (TLR2) and MyD88, which then activates protein kinase C alpha (PKCα). Through intermediates, PKCα activates ADAM-17 which cleaves the ACE2 receptor on the cell membrane producing soluble ACE2. PKCα can be inhibited by the addition of Gö 6976 and ADAM-17 can be inhibited by the addition of TAPI-1. If membrane ACE2 is intact, upon SARS-CoV-2 infection, the viral spike protein can bind the receptor and through unknown intermediates diminish IL-8 release in vitro.

Article Snippet: Eight-week-old wild-type (WT) or transgenic mice expressing the human ACE2 receptor (Jackson Labs, Bar Harbor, ME USA) were used for all animal experiments.

Techniques: In Vitro, Membrane, Infection

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: Sensing of SARS-CoV-2-infected cells by pDCs is more efficient compared with cell-free virions. ( A ) Quantification of IFN-I (IFN-α/β) in the supernatant of PBMCs that were co-cultured for 24 h with SARS-CoV-2 (LSPQ1)-infected HeLa-hACE2, exposed to cell-free SARS-CoV-2 at different MOIs, or treated with supernatant of infected cells (MOI 0.1) (sup) collected at 24 h post-infection. PBMCs used in the co-cultures and in “cell-free virus exposure” conditions were from the same donors with the same number of cells. In the latter conditions, the MOI was based on the number of PBMCs. UI, uninfected cells. Each dot represents independent experiments/donors. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Frequency of SARS-CoV-2 infection in HeLa-hACE2 as determined by flow cytometry using αN Ab and a mouse IgG1 isotype control (ctrl). (Lower panel) A summary dot plot shows the results from six independent infections. ( C ) Quantification of IFN-I in the supernatant of SARS-CoV-2-infected HeLa-hACE2 (MOI 0.1, 24 h) that were co-cultured with pDC-depleted PBMCs or total PBMCs ( n = 3 donors). ( D ) The analysis was as described for panel C but with cell-free viruses instead of infected cells ( n = 2 donors). The MOI (0.1) used was based on the number of PBMCs, as done in . In all relevant panels, the co-cultures were done for 24 h. Shown is the mean ± SEM. The dotted line indicates the limit of detection (LOD) of the assay (60 U/mL). See also .

Article Snippet: HeLa cell line stably expressing the full-length human ACE2 receptor (HeLa-hACE2) was generated by transducing HeLa cells with lentiviral vector particles expressing the human ACE2 and blasticidin-resistance genes (the lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was a kind gift from Dr. Sonja Best, Addgene plasmid #154983).

Techniques: Infection, Cell Culture, Virus, MANN-WHITNEY, Flow Cytometry, Control

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: pDC sensing of SARS-CoV-2-infected cells requires physical contact. ( A ) (Left panel) Schematic representation of the experimental design (created with BioRender.com). HeLa-hACE2 cells were infected with SARS-CoV-2 at MOI of 0.1 for 24 h and then co-cultured with PBMCs in the presence or absence of a 0.4 µm transwell membrane insert. PBMCs were added to the upper chamber of the insert. IFN-I was measured from the lower chamber, at 18–24 h after the co-cultures. (Right panel) Quantification of IFN-I in the supernatant of PBMCs co-cultured with mock- or SARS-CoV-2-infected HeLa-hACE2 (Inf cells) in the presence (+TW) or absence (–TW) of a transwell insert. Inf cells, infected cells. Data shown are mean ± SEM from independent experiments with seven donors. Each dot depicts a donor. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Design of an experiment aimed at determining whether cell-free virus can trigger residual IFN-I production in the absence of physical contact (created with BioRender.com). (Lower panel) IFN-I was measured in supernatants of PBMCs either co-cultured with infected HeLa-hACE2 (MOI 0.1) in the presence or absence of transwell inserts or exposed to cell-free virus (MOI 0.1, based on PBMC number) in the same conditions. The number of PBMCs used for the co-cultures or exposed to cell-free viruses was the same. Data shown are mean ± SEM from independent experiments with four donors. Mann-Whitney U test. * P < 0.05. In both panels: LOD, limit of detection of the IFN-I assay as described in legend. See also .

Article Snippet: HeLa cell line stably expressing the full-length human ACE2 receptor (HeLa-hACE2) was generated by transducing HeLa cells with lentiviral vector particles expressing the human ACE2 and blasticidin-resistance genes (the lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was a kind gift from Dr. Sonja Best, Addgene plasmid #154983).

Techniques: Infection, Cell Culture, Membrane, MANN-WHITNEY, Virus

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: CD54/CD11a adhesion complex is involved in the recognition and sensing of SARS-CoV-2 infected cells by pDCs in a potentially bidirectional manner ( A ) Design of a CD54 blocking experiment (created with BioRender.com). PBMCs, enriched pDCs, or SARS-CoV-2-infected cells were pretreated with various concentrations of αCD54 Ab for 30 min at room temperature before the 24 h co-cultures. Cells treated with an isotype Ab control served as a negative control. ( B ) IFN-I in the supernatant of a co-culture between infected HeLa-hACE2 and αCD54 Ab-pretreated PBMCs. The IFN-I level in the Ab-untreated condition was set at 100%, six donors. ( C ) IFN-I production in the supernatant of a 24 h co-culture whereby infected HeLa-hACE2 were pretreated with αCD54 Ab and then exposed to PBMCs. The IFN-I level in the untreated condition was set at 100%, six donors. ( D ) IFN-I release from the supernatant of SARS-CoV-2-infected Calu-3 co-cultured with anti-CD54 Ab-pretreated PBMCs. Data analysis was as described for panel B, four donors. ( E ) IFN-I from a co-culture supernatant of PBMCs with infected Calu-3 that were treated with αCD54 Ab. Data analysis was as described for Panel B, four donors. ( F ) Experimental design of a CD11a blocking experiment (created with BioRender.com). Either PBMCs were pretreated with increasing concentrations of αCD11a Ab or the αCD11a Ab was added to a co-culture of PBMCs and infected Calu-3. IFN-I was determined 18–24 h post-co-culture. ( G and H ) IFN-I production in the supernatant of infected-Calu-3 co-cultured with PBMCs. ( G ) PBMCs were pretreated with the indicated concentrations of αCD11a Ab or isotype Ab control, and IFN-I release from a co-culture with untreated PBMCs was set at 100%. ( H ) αCD11a Ab was added to the co-culture of PBMCs and infected Calu-3. IFN-I production from the Ab-untreated co-culture was set as 100%, six donors. In all relevant panels, data are shown as mean ± SEM. Statistical analysis: Mann-Whitney U test; * P < 0.05, ** P < 0.01, ns, not significant. See also .

Article Snippet: HeLa cell line stably expressing the full-length human ACE2 receptor (HeLa-hACE2) was generated by transducing HeLa cells with lentiviral vector particles expressing the human ACE2 and blasticidin-resistance genes (the lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was a kind gift from Dr. Sonja Best, Addgene plasmid #154983).

Techniques: Infection, Blocking Assay, Control, Negative Control, Co-Culture Assay, Cell Culture, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: Formation of conjugates between pDCs and SARS-CoV-2-infected cells is dependent on receptor engagement with CD54. ( A ) Schematic representation of the experimental design (created with BioRender.com). Mock- and SARS-CoV-2-infected HeLa-hACE2 were pre-labeled with cell-membrane dye eFluo 670 before co-culturing with PBMCs. Cell conjugates were analyzed by flow cytometry and defined as CD3 - CD14 - CD19 - ILT7 + BDCA2 + eFluor670 + population. Analysis was done 18–24 h after the co-culture. ( B ) (Upper panel) Dot plots showing the gating strategy to identify cell conjugates between pDCs (gated from PBMCs) and mock- or SARS-CoV-2-infected HeLa-hACE2. Numbers shown in the dot plots are cell percentages. (Lower panel) Frequency of cell conjugates from different experiments done with five donors. ( C ) (Upper panel) Representative dot plots showing the frequency of conjugates formed between enriched pDCs (CM-Dil + ) and mock- or SARS-CoV-2-infected HeLa-hACE2 (eFluor 670 + ). (Lower panel) Overall data from different experiments done with 7 PBMC donors. ( D ) (Upper panel) Representative dot plots showing the effect of blocking CD54 receptor engagement on conjugate formation between pDCs and infected HeLa-hACE2. Numbers indicated represent the percentage of conjugates. (Lower panel) Frequencies of conjugates between pDCs and eFluor670-labelled mock- and SARS-CoV-2-infected HeLa-hACE2 in the presence of αCD54 Ab or isotype control (5 µg/mL). Analysis was done with four donors. In all relevant panels, each line depicts a donor. Statistical analysis Mann-Whitney U test. * P < 0.05. See also .

Article Snippet: HeLa cell line stably expressing the full-length human ACE2 receptor (HeLa-hACE2) was generated by transducing HeLa cells with lentiviral vector particles expressing the human ACE2 and blasticidin-resistance genes (the lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was a kind gift from Dr. Sonja Best, Addgene plasmid #154983).

Techniques: Infection, Labeling, Membrane, Flow Cytometry, Co-Culture Assay, Blocking Assay, Control, MANN-WHITNEY