Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: Summary of sero-negative, acute, and convalescent-phase of COVID-19 patients from which the plasma ACE2 + EVs and RBD-IgG levels were measured.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Sampling

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of the cell-based neutralization assay. b Representative flow profiles showing the percentage (fluorescence mean intensity) of RBD-AF647 binding (at 16 and 3.3 nmol/L) to ACE2 + HEK-293 cells, inhibited by rhACE2 and ACE2 + EVs (evACE2) isolated from HEK-293 and HeLa cells (HEK-EV1 and HeLa-EV2, respectively) whereas ACE2 − EVs (evCon) had no neutralization effects (no RBD in black, PBS in dark blue, rhACE2 in orange, evCon in light blue, and evACE2 in green). c IC 50 of rhACE2 (orange line) and ACE2 in the EVs from ACE2 + HEK (ev1ACE2) and HeLa (ev2ACE2) cells (green lines) on 16 nM RBD-host cell binding (%). GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. d IC 50 of evACE2, ev1 from HEK and ev2 from HeLa cells (green lines), and rhACE2 (orange line) neutralizing infections by wild-type (WT) S + pseudotyped SARS-CoV-2. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. e IC 50 (nM) of ACE2 in ev1ACE2 (HEK) (green line) and rhACE2 (orange line) upon wild-type SARS-CoV-2 infection. GraphPad Prism 9.0.2 was used to calculate the IC 50 with three biological replicates. Data are presented as mean values ± SD. f Distinct effects of ACE2 + EVs (green lines) and ACE2 − control EVs (light blue line) on inhibiting Vero-6 cell death caused by SARS-CoV-2. N = 2 experiments with three biological replicates each. Data are presented as mean values ± SD. g The IC 50 of ev1ACE2 (HEK) neutralizing infections by pseudotyped SARS-CoV-2 expressing WT (black), B.1.1.7 (α) variant (red), B1.351 (β) variant (dark blue) and B.1.617.2 (δ) (light green) S protein. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates each. Data are presented as mean values ± SD. h Effects of ev1ACE2 (HEK) on protecting Vero-6 cell viability against infections of SARS-CoV-2 WT (black), B.1.1.7 (α) variant (red) and B1.351 (β) variant (dark blue) ( n = 3 biological replicates). Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Neutralization, Fluorescence, Binding Assay, Isolation, Infection, Control, Expressing, Variant Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of plasma EV ultracentrifugation and RBD-bead based depletion. b Cryo-EM images of human EV pellets isolated from acute phase COVID-19 plasma (bar = 100 nm). c Immunoblots of plasma EV pellets (sero-negative and COVID-19 acute phase patients CBB-005 and -013) for ACE2 and loading control of protein staining with Ponceau). Laemmli buffer was used for lysis ( N = 1 experiment). d ACE2 + EV pellets from acute phase patients 007, 008, 009, 012, and 013 (CBB) ( n = 2 biological replicates each) blocked SARS-CoV-2 infection-induced death of Vero-6 cells whereas the sero-negative control ( n = 2 biological replicates) and CBB-005 (no detectable ACE2) ( n = 2 biological replicates) did not show neutralization effects. One-tail t test, **** p = 2.24E−08 shown as compared to sero-negative. e , f Levels of ACE2 + EV counts ( n = 3 biological replicates) in plasma EVs (green) and bead-depleted EVs (light blue). One-tail paired t test, * p = 0.011 and ** p = 0.0063 (data are presented as mean values ± SD) ( e ) and altered neutralization effects on RBD–host cell binding ( f ) of the COVID-19 plasma EV pellets prior to and after RBD-bead depletion (convalescent phase CSB-012 and -024; acute phase CBB-008, 009, and 013). One-tail paired t test **** p = 5.11E−05.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Cryo-EM Sample Prep, Isolation, Western Blot, Control, Staining, Lysis, Infection, Negative Control, Neutralization, Binding Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Probability of severe disease-free survival in B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice receiving SARS-CoV-2 infection (10,000 pfu) and intranasal EVs (130 µg as measured on Nanodrop) per mouse (evCon in light blue and evACE2 in green). Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests **** p = 2.27E−07. b Viral loads in mouse lungs on day 5/6 after receiving SARS-CoV-2 infection and administration of evCon ( N = 5 mice) (light blue) or evACE2 ( N = 10 mice) (green). T -test–nonparametric-one tailed, * p = 0.013. Data are presented as mean values ± SD. c . Representative H&E images of mouse lung sections at day 5 or 6 post virus inoculation and EV treatment (evCon and evACE2) intranasally. d , e Acute and chronic inflammation scores ( d ), and alveolar hemorrhage and necrosis scores ( e ) in mouse lungs on day 5/6 after receiving evCon ( N = 5 mice) (light blue) or evACE2 ( N = 7 mice) (green). T -test–nonparametric-one tailed, ** p = 0.005, ** p = 0.003 and *** p = 0.0004. Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Infection, One-tailed Test, Virus

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