Journal: Cell Reports

Article Title: Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.108940

Figure Lengend Snippet: Berzosertib inhibits SARS-CoV-2, SARS-CoV-1, and MERS-CoV replication in human cells and is synergistic with remdesivir (A and B) Graphs show an eight-point dose-response curve of berzosertib (A) or remdesivir (B) in SARS-CoV-2-infected Calu-3 cells. Contrasted with cell viability of mock-infected cells. (C and D) Antiviral effect of berzosertib on SARS-CoV-1 (C) and MERS-CoV (D). (E) Graphs show antiviral activity measured with a SARS-CoV-2 immunostaining signal used for identification of infected A549-ACE2 cells. IC 50 values were calculated by non-linear regression sigmoidal dose-response analysis using the GraphPad Prism 7 software package. (F) Graph shows synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells. Dose-response curves obtained with mixtures of remdesivir and berzosertib, remdesivir alone (thick black line), and berzosertib alone (thick pink line) are shown. (G) Isobologram of drug combinations is depicted. (H) Combinatorial data were analyzed for inhibitory, additive, or synergistic effects (upper triangle, dotted line, and lower triangle, respectively) by using the Compusyn software package.

Article Snippet: Synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells are investigated as follows: A549-ACE2 cells were treated with a mixture of berzosertib and remdesivir (concentrations specified in the top and bottom, respectively) prepared in a 7-by-7 concentration matrix, which generated 49 combinations ranging from 0.014 μM - 10 μM for berzosertib, and 6.8 nM - 5 μM for remdesivir.

Techniques: Infection, Activity Assay, Immunostaining, Software

Journal: Cell Reports

Article Title: Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.108940

Figure Lengend Snippet:

Article Snippet: Synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells are investigated as follows: A549-ACE2 cells were treated with a mixture of berzosertib and remdesivir (concentrations specified in the top and bottom, respectively) prepared in a 7-by-7 concentration matrix, which generated 49 combinations ranging from 0.014 μM - 10 μM for berzosertib, and 6.8 nM - 5 μM for remdesivir.

Techniques: Produced, Recombinant, Electron Microscopy, Blocking Assay, MTT Cell Proliferation, Proliferation Assay, Software

Journal: bioRxiv

Article Title: SARS-CoV-2 Restructures the Host Chromatin Architecture

doi: 10.1101/2021.07.20.453146

Figure Lengend Snippet: a. A diagram showing the typical contact map patterns in Hi-C (and Hi-C 3.0 or other modified Hi-C approaches) that define A/B compartments, Topologically Associating Domains (TADs), chromatin loops or intra-TAD interactions (which perhaps include most enhancer-promoter contacts). This is an overall summary of these structures, but the exact definition of some structures may be subjected to variable interpretation, and the terminology may not always be used consistently – , . Often, A/B compartmentalization is illustrated by a checkerboard pattern of Hi-C contact matrices over large genomic sizes, indicating preferential interactions between genomic regions belonging to the same type of compartments (A: euchromatin and transcriptionally active; B: heterochromatin, transcriptionally inactive). TADs or chromatin domains are often characterized as a square or triangle-like structure on chromatin contact maps, reflecting a higher contact frequency between any regions inside the same TAD than with regions outside of the TAD. Intra-TAD enhancer-promoter contacts are considered to be facilitated by TADs, while TAD boundaries prevent aberrant interaction with regions outside of TADs. In Hi-C maps, the dot-shaped structures on the tip of domains suggests local enrichment of spatial interaction between a pair of two loci over nearby regions, and is regarded as a chromatin loop in this work. But loops may be subjected to other definitions in other studies. For example, enhancer-promoter contacts often do not appear as dot-shaped structures in Hi-C, but may be defined as loops by other work or other methods. Additional discussion, see , . b. Cartoon diagrams describe A-A and B-B association preference within regions of similar epigenetic features, which compartmentalizes chromosomes into A and B (the left part of the diagram). The diagram in the middle depicts a current model of cohesin loop extrusion inside TADs that generated such structures. The right side shows a zoom-in view of a part of a TAD that harbors enhancer-promoter contact that may play roles in gene transcriptional regulation. c. A workflow showing the experimental design. d. A barplot showing the percentage of RNA-Seq reads mapped to SARS-CoV-2 genome in Mock, 6-hr post infection (6hpi, 0.1 MOI), and 24 hpi (0.1 MOI) conditions. Mean and standard deviation (error bar) were calculated based on two biological replicates of RNA-Seq. e . Confocal images showing immunofluorescence staining of DAPI (DNA, blue) and the Spike protein of SARS-CoV-2 (red) in Mock and 24hpi (0.1 MOI) infected A549-ACE2 cells. Scale bars are shown.

Article Snippet: SARS-CoV-2 isolate USA-WA1/2020 (NR-52281; BEI Resources, Manassas, VA) was employed to infect human A549-ACE2 cells (NR53821; BEI Resources).

Techniques: Hi-C, Modification, Generated, RNA Sequencing Assay, Infection, Standard Deviation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 Restructures the Host Chromatin Architecture

doi: 10.1101/2021.07.20.453146

Figure Lengend Snippet: a. A diagram illustrating the design of spike-in calibrated ChIP-Seq using mouse ESCs as spike-in for human A549 cells (with or without infection). b,c. Barplots showing the human/mouse reads ratio in both Mock and SARS-CoV-2 conditions which permit calibrated ChIP-Seq analyses of H3K27ac, H3K4me3, H3K27me3, and H3K9me3. A scale factor for each histone mark ChIP-seq was denoted above each plot. d,e,f,g. Scatter plots show virus-caused genome-wide changes of histone mark ChIP-Seq signals at 100kb bins for H3K27ac, H3K4me3, H3K9me3 and H3K27me3. The x,y-axis are natural logarithmically scaled reads densities from calibrated ChIP-Seq data. Dotted lines denote changes by two folds. h. Snapshots of Pearson’s correlation matrices, E1 compartmental scores, sliding correlation scores (SC), as well as ChIP-Seq tracks of H3K27ac and H3K9me3 in Mock or SARS-CoV-2 infected cells. The right side shows the difference of Pearson’s correlation matrices between SARS-CoV-2 and Mock (pink shows decrease). Red arrowheads on top of the H3K27ac peaks show strong reduction of this active mark after infection, which was accompanied by quantitative increase of H3K9me3 signals at the same region (black arrowheads). Accordingly, this entire A compartment shows reduced PCA E1 scores (yellow in the E1 track), showing less compartmental interactions within the same compartment but more interactions with nearby B compartments (see the differential Pearson’s correlation matrices to the right).

Article Snippet: SARS-CoV-2 isolate USA-WA1/2020 (NR-52281; BEI Resources, Manassas, VA) was employed to infect human A549-ACE2 cells (NR53821; BEI Resources).

Techniques: ChIP-sequencing, Infection, Genome Wide

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: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Activation Assay, Lysis, Bacteria, Co-Culture Assay, Western Blot, CRISPR, Positive Control, Expressing, Microscopy, Infection, Membrane, Cell Culture, Control

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: P. aeruginosa –activated hNLRP1 inflammasome requires proteasome activity. (A) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa clinical isolates from patients with infected lung (strains 3039 1533 and 2348 4390) or with infected cornea (strain 0236 1921) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. One-way ANOVA. (B) Schematic drawing of P. aeruginosa co-culture experiments performed with human corneal epithelial cells. (C) Immunoblotting of NLRP1, Gasdermin-D, and Tubulin in pHCECs upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. Immunoblots show lysates from one experiment performed at least three times. (D) Cell lysis (LDH) and IL-1B release evaluation in pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Activity Assay, Fluorescence, Microscopy, Infection, Co-Culture Assay, Western Blot, Bacteria, Lysis

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: P. aeruginosa EEF2-inactivating EXOA promotes NLRP1 inflammasome response. (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various secretion systems (PAO1 ΔT3SS , PAO1 ΔT2SS , PAO1 ΔT1SS ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (B) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various T2SS virulence effectors (PAO1 ΔPLCN , PAO1 ΔPLCH , PAO1 ΔLASB , and PAO1 ΔEXOA ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nulcei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) or its catalytically dead mutant EXOA H426A (500 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Schematic mechanism of P. aeruginosa EXOA and related toxins at mediating EEF2 ribosylation and inactivation and subsequent ribosome inactivation. (E) Immunoblotting characterization of genetic invalidation of DPH1 in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. The red arrow shows the selected KO cells for subsequent experiments. (F) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ DPH1− reporter cell lines exposed to VbP (15 µM), EXOA (10 ng/ml), cholix toxin (CT, 10 ng/ml), and diphtheria toxin (DT, 20 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (G) Plasma membrane permeabilization determination over time using PI incorporation in WT or NLRP1 -deficient pHCECs exposed to VbP (15 µM), EXOA (10 ng/ml) or EXOA H426A (10 ng/ml) for indicated times. ***P ≤ 0.001, T test. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Microscopy, Infection, Mutagenesis, Western Blot, CRISPR, Fluorescence, Membrane

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: Multiple EEF2-targeting toxins activate the hNLRP1 inflammasome in a ZAKα-dependent manner. (A) Schematic mechanism of EXOA and related toxin-mediated translation inhibition. tRNA, transfer RNA; E, exit; P, peptidyl; A, aminoacyl. (B) Determination of ribosome inactivation in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) for 2 and 6 h by measuring ribosome polysome accumulation and puromycin incorporation. Images are representative of one experiment performed at least three times. (C) Immunoblotting of ADP-ribosylated proteins, EEF2, and Tubulin in A549 NLRP1+/ASC-GFP cell lysates treated or not with VbP (15 µM) or EXOA (10 ng/ml) in the presence of Nicotinamide adenine dinucleotide-Biotin (NAD-Biot). Immunoblots show lysates from one experiment performed at least three times. (D) Immunoblotting of NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1− reporter cell lines exposed or not to EXOA (10 ng/ml) or its inactive mutant EXOA H426A for 3 h. Immunoblots show lysates from one experiment performed at least three times. (E) Immunoblotting characterization of genetic invalidation of P38α and P38β in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. Immunoblots show lysates from one experiment performed at least three times. (F) Fluorescence microscopy of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ ZAKα - reporter cell lines expressing hACE2 infected for 24 h with various SARS-CoV-2 MOI. ASC-GFP (green) pictures were taken in the dish after viral infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. Source data are available for this figure: .

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Inhibition, Western Blot, TNKS1 Histone Ribosylation Assay, Mutagenesis, CRISPR, Fluorescence, Microscopy, Expressing, Infection

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: EEF2 inactivation drives ZAKα and P38 MAPK activation and subsequent NLRP1 inflammasome nucleation. (A) Immunoblotting of P38, JNK, ZAKα, NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1+/ZAKα- reporter cell lines exposed or not to EXOA (10 ng/ml) for 3 h. Immunoblots show lysates from one experiment performed at least three times. (B) Cell lysis (LDH release), florescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ZAKα- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positives for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. (C) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/P38α/β- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicate at least three times. (D) Western blot examination of NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 4 h exposure to EXOA (10 ng/ml) or VbP (15 µM). Images shown are from one experiment and are representative of n = 3 independent experiments. (E) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 10 h exposure to EXOA (10 ng/ml) or VbP (15 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available for this figure: .

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Activation Assay, Western Blot, Lysis, Microscopy, Fluorescence, Plasmid Preparation, Construct

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: List of reagents used in the study

Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

Techniques: Protease Inhibitor, Staining, Marker, CyQUANT Assay, Mutagenesis, Membrane, Recombinant