Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Western blot showing the expression of E1B-55K target genes in A549 cells infected with either wildtype (HA-tagged E1B-55K-expressing) virus or a ΔE1B-55K mutant (lacking E1B-55K expression). Cells were infected with MOI 30 and analyzed after 24 hours. This representative replicate illustrates the steady-state levels of p53, Mre11 and CDKN1A during infection (antibodies are listed in ). (B) Principal component analysis of mock-, wildtype- and ΔE1B-55K-infected A549 cells after differential gene expression analysis. (C) Volcano plot comparing gene expression between wildtype- and ΔE1B-55K-infected A549 cells. Genes with an adjusted P -value < 0.1 and a log₂ fold change < -1 are shown in blue, while those with an adjusted P -value < 0.1 and a log₂ fold change > 1 are shown in red. Genes belonging to the p53 transcriptional network (WikiPathways database) are highlighted with green circles. (D) Heatmap displaying row Z-scores of p53 pathway genes that were downregulated in (C) and are marked in green. The grey, black, and purple boxes at the top of the panel represent mock, wildtype, and ΔE1B-55K infection, respectively. (E) Global pathway analysis of differentially expressed genes comparing wildtype- with ΔE1B-55K-infected A549 cells using gene set enrichment analysis. The normalized enrichment scores of significantly up- and downregulated pathways are shown, with pathways meeting FDR < 0.1 highlighted in light blue/red and those with FDR < 0.05 in dark blue/red. p53-associated pathways are written in green. Red bars indicate pathways enriched in upregulated genes, while blue bars indicate pathways enriched in downregulated genes.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Western Blot, Expressing, Infection, Virus, Mutagenesis, Gene Expression

Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Interferon-alpha induction scheme in A549 and H1299 cells. The time points post-infection at which DNA is extracted for viral DNA quantification are illustrated below the timeline bar. (B) Western blot from one of the sequenced replicates, displaying selected viral- and IFN-induced proteins (antibodies are listed in ). Proteins associated with the p53 pathway and the immune response are marked in green and pink, respectively. The upper seven proteins were detected on medical X-ray films and the lower five proteins were visualized via the ChemoStar Plus. Additional replicates are shown in . (C) Virus yield was determined 24 hpi with MOI 30 by anti-DBP (B6-8) immunofluorescence staining. Bar graphs represent the mean virus yield of three independent experiments, error bars indicate SD. Black and purple bars represent wildtype and ∆E1B-55K virus, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively. Statistical significance was determined using two-tailed t-test. ** P -value < 0.01, * P -value < 0.05, ns P -value > 0.05. (D) Normalized viral mRNA counts of selected early (upper row) and late (lower row) genes. *adjusted P -value < 0.1; ns adjusted P -value > 0.1. Light grey, black, and purple box plots represent mock, wildtype virus, and ΔE1B-55K virus conditions, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Infection, Western Blot, Virus, Immunofluorescence, Staining, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Adenovirus E1B-55K regulates p53-dependent and -independent gene expression during infection

doi: 10.1371/journal.ppat.1013622

Figure Lengend Snippet: (A) Principal component analysis of mock-, wildtype- and ΔE1B-55K-infected A549 (left) and H1299 (right) cells after differential gene expression analysis. Circles and triangles indicate BSA and IFN treatment, respectively. (B) Pathway analysis of downregulated genes comparing wildtype with ΔE1B-55K infection upon IFN treatment using GSEA, showing the normalized enrichment score for pathways with FDR < 0.1 (light blue) and FDR < 0.05 (dark blue). The upper and lower bar graphs represent pathways enriched in infected A549 and H1299 cells, respectively. Only downregulated pathways are displayed; upregulated pathways are shown in and . Immune response-associated pathways are highlighted in pink and p53-associated pathways in green. The dotted line separates the two cell lines, as labeled on the right. (C) Normalized viral mRNA counts of selected p53 and immune-system associated pathways from wildtype- and ΔE1B-55K-infected A549 (top) and H1299 (bottom) cells treated with IFN. ****adjusted P -value < 0.0001, ***adjusted P -value < 0.001, *adjusted P -value < 0.1, ns adjusted P -value > 0.1. Light grey, black, and purple box plots represent mock, wildtype virus, and ΔE1B-55K virus conditions, respectively. Treatment with BSA or IFN is indicated with white and pink background, respectively. (D) Volcano plot comparing gene expression between wildtype- and ΔE1B-55K-infected A549 (top) and H1299 (bottom) cells treated with IFN. Genes with an adjusted P -value < 0.1 and log 2 fold change < 0 are colored in blue, while genes with an adjusted P -value < 0.1 and log 2 fold change > 0 were colored in red. Genes that belong to the IFN-associated gene network are shown with pink dots.

Article Snippet: A549 (DMSZ ACC 107; German Collection of Microorganisms and Cell Cultures), H1299 (ATCC CRL-5803; American Type Culture Collection) and primary human foreskin fibroblasts (HFF) were maintained as monolayers in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin solution (10,000 U/ml penicillin; 10 mg/ml streptomycin in 0.9% NaCl, PAN Biotech) at 37°C and 5% CO 2 .

Techniques: Infection, Gene Expression, Labeling, Virus

Journal: PLoS ONE

Article Title: The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection

doi: 10.1371/journal.pone.0126437

Figure Lengend Snippet: Panel A : E . coli strains were incubated with epithelial cells for 30 min at 37°C. Following this, cells were washed to remove unbound bacteria, lysed, diluted, and spread onto agar plates to calculate the number of bound bacteria. The results are expressed as the mean percentage (± standard error) of inoculated bacteria attached to A549 cells. The values in parentheses show the actual percentage. Panel B : E . coli strains were cultured in the wells of PVC microplates, stained with crystal violet, washed with deionized water, and the wells were photographed. The arrow shows biofilm formation, which was quantitated by extracting crystal violet with methanol and measuring absorbance at 570 nm. The results are shown in parentheses and are expressed as the mean (± standard error) absorbance. Both panels : The asterisks indicate that the increase in adherence and biofilm formation of E . coli carrying pBpaB, compared to E . coli harboring pBHR1ΔDra, is statistically significant ( P values < 0.05, Mann-Whitney test).

Article Snippet: The cell lines A549 (human type II alveolar epithelium; ATCC CCL85) and J774A.1 (murine macrophages; ATCC TIB-67) were cultured as described elsewhere [ , ].

Techniques: Incubation, Bacteria, Cell Culture, Staining, MANN-WHITNEY

Journal: Pharmaceuticals

Article Title: Improved HDAC Inhibition, Stronger Cytotoxic Effect and Higher Selectivity against Leukemias and Lymphomas of Novel, Tricyclic Vorinostat Analogues

doi: 10.3390/ph14090851

Figure Lengend Snippet: IC 50 [µM] of Vorinostat derivatives 7a-t based on the survival of non-cancerous (BALB/3T3) and cancerous (MV4-11, Daudi, MCF-7 and A549) cells after 72 h of treatment. N/T—not tested.

Article Snippet: Human biphenotypic B myelomonocytic leukemia MV4-11 and normal mouse fibroblast BALB/3T3 cell line were obtained from American Type Culture Collection (USA); human lung carcinoma A549 cell line and human adenocarcinoma breast cancer MCF-7 cell line were obtained from European Collection of Authenticated Cell Cultures (UK).

Techniques:

Journal: Pharmaceuticals

Article Title: Improved HDAC Inhibition, Stronger Cytotoxic Effect and Higher Selectivity against Leukemias and Lymphomas of Novel, Tricyclic Vorinostat Analogues

doi: 10.3390/ph14090851

Figure Lengend Snippet: Selectivity index (IC 50 of normal vs. cancer cells). SI > 1.0 indicates a compound of greater activity against cancer cells and lower cytotoxicity on normal cells.

Article Snippet: Human biphenotypic B myelomonocytic leukemia MV4-11 and normal mouse fibroblast BALB/3T3 cell line were obtained from American Type Culture Collection (USA); human lung carcinoma A549 cell line and human adenocarcinoma breast cancer MCF-7 cell line were obtained from European Collection of Authenticated Cell Cultures (UK).

Techniques: Activity Assay

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 Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B

doi: 10.4196/kjpp.2023.27.2.131

Figure Lengend Snippet: (A) RT-qPCR analysis results of circ_0129047 in both LUAD and normal tissues. (B) RT-qPCR analysis of circ_0129047 in human bronchial epithelium (BEAS-2B) and LUAD cells (A549, H1975, PC9 and Calu-3). **p < 0.01 vs . BEAS-2B. (C) Loop structures of Circ_012904. (D) Subcellular localization of circ_0129047 in PC9 and Calu-3 cells. (E) Circ_012904 was resistant to RNase R digestion. **p < 0.01 vs . RNAse R. Values are presented as mean ± SD.

Article Snippet: Human bronchial epithelium (BEAS-2B, cat#: CRL-9609), LUAD cells (A549, cat#: CRM-CCL-185 derived from the lung tissue of a white, 58-year-old male with LUAD; H1975, CRL-5908 derived from the lung tissue of a nonsmoking female with LUAD; and Calu-3, cat#: HTB-55, derived from the lung tissue of a white, 25-year-old male with LUAD) were obtained from the American Type Culture Collection (ATCC), and PC9 (cat#: 90071810, derived from the lung tissue of an undifferentiated patient with LUAD) was obtained from Sigma-Aldrich.

Techniques: Quantitative RT-PCR

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B

doi: 10.4196/kjpp.2023.27.2.131

Figure Lengend Snippet: (A) MiR-665 is predicted as a target of circ_0129047 through CircInteractome. (B) RIP assay was performed using Ago2 antibody in LUAD cells, and the enrichment of circ_0129047 and miR-665 was detected. **p < 0.01 vs . Anti-IgG. (C) Luciferase activity of Circ_0129047 in LUAD cells transfected with miR-665 mimics, which bind to the circ 0129047 sequence. **p < 0.01 vs . mimic-NC. (D) RT-qPCR analysis of miR-665 in LUAD tissues and normal tissues. (E) RT-qPCR analysis of miR-665 in LUAD cells (PC9 and Calu-3) and normal cells (BEAS-2B). **p < 0.01 vs . BEAS-2B. (F) Pearson analysis of miR-665 expression and circ_0129047 expression in LUAD tissues. (G) circ 0129047-overexpressing vectors (OE-circ), OE-NC, miR-665 mimic (mimic), mimic-NC, and OE+mimic were transfected into PC9 and Calu-3 cells. The expression of miR-665 was measured using RT-qPCR 48 h after transfection. **p < 0.01 vs . OE-NC; ## p < 0.01 vs . mimic-NC; && p < 0.01 vs . OE+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; Ago2, Argonaute 2; WT, wild type; MUT, mutant.

Article Snippet: Human bronchial epithelium (BEAS-2B, cat#: CRL-9609), LUAD cells (A549, cat#: CRM-CCL-185 derived from the lung tissue of a white, 58-year-old male with LUAD; H1975, CRL-5908 derived from the lung tissue of a nonsmoking female with LUAD; and Calu-3, cat#: HTB-55, derived from the lung tissue of a white, 25-year-old male with LUAD) were obtained from the American Type Culture Collection (ATCC), and PC9 (cat#: 90071810, derived from the lung tissue of an undifferentiated patient with LUAD) was obtained from Sigma-Aldrich.

Techniques: Luciferase, Activity Assay, Transfection, Sequencing, Quantitative RT-PCR, Expressing, Mutagenesis

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B

doi: 10.4196/kjpp.2023.27.2.131

Figure Lengend Snippet: (A) Predicted miR-665 interactions with PTPRB 3′UTR using miRNA target prediction software based on TargetScan. (B) Luciferase reporter gene assay of PTPRB 3′UTR after miR-665 treatment. **p < 0.01 vs . mimic-NC. (C) PTPRB mRNA expression in LUAD tissues analyzed using RT-qPCR. (D) Pearson correlation analysis was used to analyze the relationship between miR-665 and PTPRB in LUAD tissues. (E) Pearson correlation analysis was used to analyze the relationship between circ_0129047 and PTPRB in LUAD tissues. (F) PTPRB mRNA expression in LUAD cells (PC9 and Calu-3) and normal cells (BEAS-2B) using RT-qPCR. **p < 0.01 vs . BEAS-2B. (G) Western blot analysis determining PTPRB expression in LUAD cells transfected with PTPRB-overexpressing vectors (OE-PTPRB), OE-NC, miR-665 mimic (mimic), mimic-NC, OE-PTPRB+mimic. **p < 0.01 vs . OE-NC; ## p < 0.01 vs . mimic-NC; && p < 0.01 vs . OE-PTPRB+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; PTPRB, protein tyrosine phosphatase receptor type B; WT, wild type; MUT, mutant.

Article Snippet: Human bronchial epithelium (BEAS-2B, cat#: CRL-9609), LUAD cells (A549, cat#: CRM-CCL-185 derived from the lung tissue of a white, 58-year-old male with LUAD; H1975, CRL-5908 derived from the lung tissue of a nonsmoking female with LUAD; and Calu-3, cat#: HTB-55, derived from the lung tissue of a white, 25-year-old male with LUAD) were obtained from the American Type Culture Collection (ATCC), and PC9 (cat#: 90071810, derived from the lung tissue of an undifferentiated patient with LUAD) was obtained from Sigma-Aldrich.

Techniques: Software, Luciferase, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Mutagenesis

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B

doi: 10.4196/kjpp.2023.27.2.131

Figure Lengend Snippet: LUAD cells were transfected with PTPRB-overexpressing vectors (OE-PTPRB), OE-NC, miR-665 mimic (mimic), mimic-NC, and OE-PTPRB+mimic. (A) Cell proliferation was analyzed using the CCK-8 assay. (B) Western blot analysis of anti-Bax and anti-Bcl-2 expression in the transfected cells. (C) Colony formation was determined using colony formation assay (×200). *p < 0.05, **p < 0.01 vs . OE-NC; # p < 0.05, ## p < 0.01 vs . mimic-NC; & p < 0.05, && p < 0.01 vs . OE-PTPRB+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; OD, optical density; PTPRB, protein tyrosine phosphatase receptor type B.

Article Snippet: Human bronchial epithelium (BEAS-2B, cat#: CRL-9609), LUAD cells (A549, cat#: CRM-CCL-185 derived from the lung tissue of a white, 58-year-old male with LUAD; H1975, CRL-5908 derived from the lung tissue of a nonsmoking female with LUAD; and Calu-3, cat#: HTB-55, derived from the lung tissue of a white, 25-year-old male with LUAD) were obtained from the American Type Culture Collection (ATCC), and PC9 (cat#: 90071810, derived from the lung tissue of an undifferentiated patient with LUAD) was obtained from Sigma-Aldrich.

Techniques: Transfection, CCK-8 Assay, Western Blot, Expressing, Colony Assay