vero cells  (Thermo Fisher)


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    Name:
    VP SFM 1X
    Description:
    VP SFM is a serum free ultra low protein 5 µg ml medium containing no proteins peptides or other components of animal or human origin It is designed for the growth of VERO cells for virus production 1 This medium is suitable for the growth of COS 7 MDCK BHK 21 suspension culture and HEp2 cells and for recombinant protein production Formulated without L glutamine for increased stability add 20 ml of 200 mM L glutamine or 20 ml of GlutaMAX I supplement per 1 000 ml of medium prior to use
    Catalog Number:
    11681020
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Bioproduction|Cell Culture|Mammalian Cell Culture|Vaccine & Virus Production|Viral Vaccine Production
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    Structured Review

    Thermo Fisher vero cells
    Digital images of confocal micrographs showing <t>UL11</t> localization. <t>Vero</t> cell monolayers were transfected with the UL11-GFP-expressing plasmid. At 24 h posttransfection, cells were washed thoroughly, fixed, and stained with the anti-GFP antibody (A1) or
    VP SFM is a serum free ultra low protein 5 µg ml medium containing no proteins peptides or other components of animal or human origin It is designed for the growth of VERO cells for virus production 1 This medium is suitable for the growth of COS 7 MDCK BHK 21 suspension culture and HEp2 cells and for recombinant protein production Formulated without L glutamine for increased stability add 20 ml of 200 mM L glutamine or 20 ml of GlutaMAX I supplement per 1 000 ml of medium prior to use
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    Price from $9.99 to $1999.99
    vero cells - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "UL20 Protein Functions Precede and Are Required for the UL11 Functions of Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment ▿"

    Article Title: UL20 Protein Functions Precede and Are Required for the UL11 Functions of Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02201-06

    Digital images of confocal micrographs showing UL11 localization. Vero cell monolayers were transfected with the UL11-GFP-expressing plasmid. At 24 h posttransfection, cells were washed thoroughly, fixed, and stained with the anti-GFP antibody (A1) or
    Figure Legend Snippet: Digital images of confocal micrographs showing UL11 localization. Vero cell monolayers were transfected with the UL11-GFP-expressing plasmid. At 24 h posttransfection, cells were washed thoroughly, fixed, and stained with the anti-GFP antibody (A1) or

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining

    Intracellular localization of the UL11 and UL20 proteins in virus-infected cells. Vero cells were transfected with plasmids expressing UL11-GFP, UL20, or both. At 24 h posttransfection, cells were infected at an MOI of 2 with YEbac102ΔUL11ΔUL20.
    Figure Legend Snippet: Intracellular localization of the UL11 and UL20 proteins in virus-infected cells. Vero cells were transfected with plasmids expressing UL11-GFP, UL20, or both. At 24 h posttransfection, cells were infected at an MOI of 2 with YEbac102ΔUL11ΔUL20.

    Techniques Used: Infection, Transfection, Expressing

    Plaque phenotypes of UL11-null, UL20-null, and UL11/UL20 double-null viruses under complementing and noncomplementing conditions. Vero cell monolayers were either mock transfected or transfected with plasmids expressing UL11, UL20, or both UL11 and UL20.
    Figure Legend Snippet: Plaque phenotypes of UL11-null, UL20-null, and UL11/UL20 double-null viruses under complementing and noncomplementing conditions. Vero cell monolayers were either mock transfected or transfected with plasmids expressing UL11, UL20, or both UL11 and UL20.

    Techniques Used: Transfection, Expressing

    Comparison of UL11 intracellular localization in the presence or absence of gK/UL20. Vero cells were transfected with a combination of plasmids expressing UL11-GFP, UL20, and/or gK. At 24 h posttransfection, cells were washed thoroughly, fixed, and prepared
    Figure Legend Snippet: Comparison of UL11 intracellular localization in the presence or absence of gK/UL20. Vero cells were transfected with a combination of plasmids expressing UL11-GFP, UL20, and/or gK. At 24 h posttransfection, cells were washed thoroughly, fixed, and prepared

    Techniques Used: Transfection, Expressing

    2) Product Images from "Expression of the Rickettsia prowazekii pld or tlyC Gene in Salmonella enterica Serovar Typhimurium Mediates Phagosomal Escape "

    Article Title: Expression of the Rickettsia prowazekii pld or tlyC Gene in Salmonella enterica Serovar Typhimurium Mediates Phagosomal Escape

    Journal:

    doi: 10.1128/IAI.73.10.6668-6673.2005

    mRNA expression of four potentially membranolytic genes of Rickettsia prowazekii in Vero cell culture at 30 min postinfection. (A) RT-PCR using primers specific for tlyA , pat1 , tlyC , and pld . (B) PCRs using R. prowazekii DNA demonstrated the effectiveness
    Figure Legend Snippet: mRNA expression of four potentially membranolytic genes of Rickettsia prowazekii in Vero cell culture at 30 min postinfection. (A) RT-PCR using primers specific for tlyA , pat1 , tlyC , and pld . (B) PCRs using R. prowazekii DNA demonstrated the effectiveness

    Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Proportions of R. prowazekii organisms within Vero cell cytosol at 30 and 50 min postinfection. The difference between proportions at 30 min and 50 min postinfection was statistically significant ( P
    Figure Legend Snippet: Proportions of R. prowazekii organisms within Vero cell cytosol at 30 and 50 min postinfection. The difference between proportions at 30 min and 50 min postinfection was statistically significant ( P

    Techniques Used:

    3) Product Images from "Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures"

    Article Title: Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.13.7467-7476.2003

    Reactivity of ICP8 with antibodies as a function of chase time. Vero cells were infected with WT KOS1.1 virus at an MOI of 20 and pulse-labeled with [ 35 S]methionine for 3 min at 4.5 hpi. At the indicated times after labeling, infected cells were harvested, and ICP8 was immunoprecipitated from aliquots of each lysate, using the conformation-dependent MAb 39S or the polyclonal antiserum R219, which recognizes the N terminus of ICP8. Immune complexes were resolved by SDS-PAGE and quantified with a phosphorimager. The amounts immunoprecipitated are expressed as a percentage of the total ICP8 in each sample.
    Figure Legend Snippet: Reactivity of ICP8 with antibodies as a function of chase time. Vero cells were infected with WT KOS1.1 virus at an MOI of 20 and pulse-labeled with [ 35 S]methionine for 3 min at 4.5 hpi. At the indicated times after labeling, infected cells were harvested, and ICP8 was immunoprecipitated from aliquots of each lysate, using the conformation-dependent MAb 39S or the polyclonal antiserum R219, which recognizes the N terminus of ICP8. Immune complexes were resolved by SDS-PAGE and quantified with a phosphorimager. The amounts immunoprecipitated are expressed as a percentage of the total ICP8 in each sample.

    Techniques Used: Infection, Labeling, Immunoprecipitation, SDS Page

    4) Product Images from "Characterization of SARS‐CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence‐based assay), Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay"

    Article Title: Characterization of SARS‐CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence‐based assay), Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay

    Journal: Febs Letters

    doi: 10.1016/j.febslet.2004.09.026

    Anti‐SARS‐CoV activities of MP576 and MP521. (A) Vero cells infected with 100 TCID 50 SARS‐CoV. (B) Vero cells infected with 100 TCID 50 SARS‐CoV in the presence of 20 μg/ml of MP576. (C) Vero cells infected with 100 TCID 50 SARS‐CoV in the presence of 20 μg/ml of MP521. CPE were recorded 96 h post infection. Experiments were carried out in duplicate and repeated twice.
    Figure Legend Snippet: Anti‐SARS‐CoV activities of MP576 and MP521. (A) Vero cells infected with 100 TCID 50 SARS‐CoV. (B) Vero cells infected with 100 TCID 50 SARS‐CoV in the presence of 20 μg/ml of MP576. (C) Vero cells infected with 100 TCID 50 SARS‐CoV in the presence of 20 μg/ml of MP521. CPE were recorded 96 h post infection. Experiments were carried out in duplicate and repeated twice.

    Techniques Used: Infection

    5) Product Images from "Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication"

    Article Title: Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication

    Journal: Viruses

    doi: 10.3390/v12101093

    TG priming enhanced type I/III IFN-dependent gene expression. ( A – G ) NHBE cells were incubated for 30 min with DMSO or 0.01 µM TG and subsequently infected with USSR H1N1 virus at 0.5 MOI for 24 h. Total RNA was extracted from each sample for type I/III IFN and indicated associated gene expression, normalised to 18S rRNA. Significance determined by two-way ANOVA, relative to corresponding DMSO control. ( H ) Vero cells were primed with TG as indicated for 30 min, washed twice with PBS and infected with USSR virus 0.5 MOI. Viral RNA extraction was performed on culture media at 24 and 48 hpi followed by one-step reverse transcription qPCR to detect the relative copy number of M-gene RNA, based on relative Ct method. Vero cells are unable to produce type I IFNs, which appear necessary for TG to induce an antiviral state. Indicated significance based 2-way RM ANOVA relative to corresponding DMSO control. *** p
    Figure Legend Snippet: TG priming enhanced type I/III IFN-dependent gene expression. ( A – G ) NHBE cells were incubated for 30 min with DMSO or 0.01 µM TG and subsequently infected with USSR H1N1 virus at 0.5 MOI for 24 h. Total RNA was extracted from each sample for type I/III IFN and indicated associated gene expression, normalised to 18S rRNA. Significance determined by two-way ANOVA, relative to corresponding DMSO control. ( H ) Vero cells were primed with TG as indicated for 30 min, washed twice with PBS and infected with USSR virus 0.5 MOI. Viral RNA extraction was performed on culture media at 24 and 48 hpi followed by one-step reverse transcription qPCR to detect the relative copy number of M-gene RNA, based on relative Ct method. Vero cells are unable to produce type I IFNs, which appear necessary for TG to induce an antiviral state. Indicated significance based 2-way RM ANOVA relative to corresponding DMSO control. *** p

    Techniques Used: Expressing, Incubation, Infection, RNA Extraction, Real-time Polymerase Chain Reaction

    6) Product Images from "?-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-?B signaling"

    Article Title: ?-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-?B signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-12-29

    The ISG promoter activity is triggered by β- and γ-catenin. (A) A549 cells were transfected with β-catenin and LEF1 for 30 h, and the mRNA level of the type I and type III IFN-dependent MX1 gene was measured by qRT-PCR. The mRNA amount of empty vector-transfected cells was taken as unity. (B and C) Vero cells transfected for 24 h with indicated plasmids were infected with vesicular stomatitis virus (VSV) (MOI = 0.0001) for an additional 24 h. Subsequently, the overexpression of β-catenin was confirmed by immunoblotting of corresponding RIPA lysates (B) and the propagation of VSV by standard plaque titration assay (C) . (D and E) Vero cells were co-transfected with the ISRE luciferase reporter gene and plasmids coding for proteins indicated in column legends. After 24 hours, Vero cells were left unstimulated or treated with 100 U/ml IFN-β for 8 h. The y-axis represents the relative reporter gene activity with luciferase activity of unstimulated, empty vector-transfected cells being set to one. (F) Vero cells were transfected with the ISRE luciferase reporter gene, and its activity in β-catenin- and LEF1-overexpressing cells was measured in the presence or absence of co-transfected p300. The luciferase activity of β-catenin and LEF1-transfected cells was arbitrarily taken as unity. (G) A549 cells were transfected with empty vector or plasmids coding for β-catenin and LEF1 for 30 h, and the interaction of cellular proteins with the DNA was analyzed by ChIP assays using specific antibodies to IRF3 or β-catenin. The co-immunoprecipitated DNA was amplified by qRT-PCR using specific primers for the promoter region of the MX1 gene and is given as the n-fold amount to the IgG control. Representative values from one of three repeated experiments are depicted.
    Figure Legend Snippet: The ISG promoter activity is triggered by β- and γ-catenin. (A) A549 cells were transfected with β-catenin and LEF1 for 30 h, and the mRNA level of the type I and type III IFN-dependent MX1 gene was measured by qRT-PCR. The mRNA amount of empty vector-transfected cells was taken as unity. (B and C) Vero cells transfected for 24 h with indicated plasmids were infected with vesicular stomatitis virus (VSV) (MOI = 0.0001) for an additional 24 h. Subsequently, the overexpression of β-catenin was confirmed by immunoblotting of corresponding RIPA lysates (B) and the propagation of VSV by standard plaque titration assay (C) . (D and E) Vero cells were co-transfected with the ISRE luciferase reporter gene and plasmids coding for proteins indicated in column legends. After 24 hours, Vero cells were left unstimulated or treated with 100 U/ml IFN-β for 8 h. The y-axis represents the relative reporter gene activity with luciferase activity of unstimulated, empty vector-transfected cells being set to one. (F) Vero cells were transfected with the ISRE luciferase reporter gene, and its activity in β-catenin- and LEF1-overexpressing cells was measured in the presence or absence of co-transfected p300. The luciferase activity of β-catenin and LEF1-transfected cells was arbitrarily taken as unity. (G) A549 cells were transfected with empty vector or plasmids coding for β-catenin and LEF1 for 30 h, and the interaction of cellular proteins with the DNA was analyzed by ChIP assays using specific antibodies to IRF3 or β-catenin. The co-immunoprecipitated DNA was amplified by qRT-PCR using specific primers for the promoter region of the MX1 gene and is given as the n-fold amount to the IgG control. Representative values from one of three repeated experiments are depicted.

    Techniques Used: Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, Infection, Over Expression, Titration, Luciferase, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification

    Regulation of the IFN-β promoter by catenins and LEF1. (A - D) A549 or Vero cells were transfected with IFN-β luciferase gene reporter plasmid (indicated above the images) along with plasmids specified in column legends for 24 h and either left untreated or stimulated (by transfection) for 5 h with 500 ng of cellular RNA (isolated from uninfected A549 cells), viral RNA (isolated from PR8-infected A549 cells) or 1 μg of 5′-triphosphate modified RNA (pppRNA) per well. Luciferase activity of unstimulated or cellular RNA-stimulated and empty vector-transfected cells was taken as unity. (E) A549 cells were transfected with indicated plasmids for 24 h and conditioned media (donor cells) were harvested before the cells were lysed. The RIPA cell lysates were then analyzed for the efficacy of transfection by Western blotting (left panel). The conditioned media were applied to freshly plated A549 cells (acceptor cells) for 15 min and cells stimulated with conditioned media were then analyzed for phosphorylation of STAT1 at Y701 (right panel). Immunoblotting of β-actin served as loading controls. (F - H) Vero cells were transfected with luciferase reporter plasmids containing either the IRF3- (F) , AP-1- (G) or NF-κB- (H) dependent binding region of the IFN-β enhanceosome along with plasmids indicated in column legends. The activity of reporter genes was determined 30 h after transfection. Luciferase activity of empty vector-transfected cells was always taken as unity. Mean values from three independent experiments are depicted in A , D , F , G and H , whereas B , C and E show representative images of three independent experiments.
    Figure Legend Snippet: Regulation of the IFN-β promoter by catenins and LEF1. (A - D) A549 or Vero cells were transfected with IFN-β luciferase gene reporter plasmid (indicated above the images) along with plasmids specified in column legends for 24 h and either left untreated or stimulated (by transfection) for 5 h with 500 ng of cellular RNA (isolated from uninfected A549 cells), viral RNA (isolated from PR8-infected A549 cells) or 1 μg of 5′-triphosphate modified RNA (pppRNA) per well. Luciferase activity of unstimulated or cellular RNA-stimulated and empty vector-transfected cells was taken as unity. (E) A549 cells were transfected with indicated plasmids for 24 h and conditioned media (donor cells) were harvested before the cells were lysed. The RIPA cell lysates were then analyzed for the efficacy of transfection by Western blotting (left panel). The conditioned media were applied to freshly plated A549 cells (acceptor cells) for 15 min and cells stimulated with conditioned media were then analyzed for phosphorylation of STAT1 at Y701 (right panel). Immunoblotting of β-actin served as loading controls. (F - H) Vero cells were transfected with luciferase reporter plasmids containing either the IRF3- (F) , AP-1- (G) or NF-κB- (H) dependent binding region of the IFN-β enhanceosome along with plasmids indicated in column legends. The activity of reporter genes was determined 30 h after transfection. Luciferase activity of empty vector-transfected cells was always taken as unity. Mean values from three independent experiments are depicted in A , D , F , G and H , whereas B , C and E show representative images of three independent experiments.

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Isolation, Infection, Modification, Activity Assay, Western Blot, Binding Assay

    β-catenin and LEF1 regulate IFN-β promoter activity via IRF-PRD. (A) Vero cells were transfected with the reporter gene plasmid containing the IRF3-responsive elements of the IFN-β enhanceosome along with indicated plasmids, and the reporter gene activity was measured 30 h later. The luciferase activity of β-catenin and LEF1-transfected cells was arbitrarily taken as unity. (B) A549 cells were transfected with empty vector or plasmids coding for β-catenin and LEF1 for 30 h, and the interaction of cellular proteins with the DNA was analyzed by ChIP assays. The amount of amplified DNA in IRF3- and β-catenin-specific immunoprecipitates was quantified by qRT-PCR using primers specific for the promoter region of the IFNB1 gene. Values represent n-folds of IgG controls. One of three independently repeated experiments is depicted as representative.
    Figure Legend Snippet: β-catenin and LEF1 regulate IFN-β promoter activity via IRF-PRD. (A) Vero cells were transfected with the reporter gene plasmid containing the IRF3-responsive elements of the IFN-β enhanceosome along with indicated plasmids, and the reporter gene activity was measured 30 h later. The luciferase activity of β-catenin and LEF1-transfected cells was arbitrarily taken as unity. (B) A549 cells were transfected with empty vector or plasmids coding for β-catenin and LEF1 for 30 h, and the interaction of cellular proteins with the DNA was analyzed by ChIP assays. The amount of amplified DNA in IRF3- and β-catenin-specific immunoprecipitates was quantified by qRT-PCR using primers specific for the promoter region of the IFNB1 gene. Values represent n-folds of IgG controls. One of three independently repeated experiments is depicted as representative.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR

    IAV infection inhibits β-catenin-mediated transcriptional activation of LEF/TCF-dependent target genes. (A and B) A549 cells were transfected with the TopFlash reporter construct together with an empty vector or a plasmid coding for β-catenin. At 24 h post transfection, the cells were infected with FPV (MOI = 5) or stimulated (via transfection) for an additional 8 h with 500 ng of cellular or viral RNA or left untreated. Subsequently, the promoter activity was measured. The luciferase activity of mock-infected or unstimulated but empty vector-transfected cells was taken as unity. (C) A schedule of signaling cascades activated by IAV RNA is depicted (adapted from [ 10 , 55 , 56 ]). (D and E) HEK293 cells were transfected with the TopFlash reporter gene construct together with β-catenin and plasmids coding for the indicated proteins. At 30 h post transfection, the promoter activity was measured (D) . The luciferase activity of β-catenin and empty vector-transfected cells was arbitrarily taken as unity. Overexpression of the recombinant proteins was analyzed by Western blotting. The β-actin immunoblots always served as loading controls (E) . (F) A549 cells transfected with the TopFlash reporter plasmid together with β-catenin were stimulated with 5 mM of BAY inhibitor or DMSO as control. At 16 h post stimulation, cells were transfected with 500 ng cellular or viral RNA for an additional 8 h and the promoter activity was measured. The luciferase activity of cells stimulated with cellular RNA was always taken as unity. (G and H) Vero cells were transfected with reporter gene plasmids containing either the IRF3 responsive elements of the IFN-β enhanceosome (G) or the ISRE motif (H) along with plasmids coding for proteins indicated in column legends. The promoter activity was measured 30 h post transfection. Luciferase activity of cells transfected with any vector control was arbitrarily taken as unity.
    Figure Legend Snippet: IAV infection inhibits β-catenin-mediated transcriptional activation of LEF/TCF-dependent target genes. (A and B) A549 cells were transfected with the TopFlash reporter construct together with an empty vector or a plasmid coding for β-catenin. At 24 h post transfection, the cells were infected with FPV (MOI = 5) or stimulated (via transfection) for an additional 8 h with 500 ng of cellular or viral RNA or left untreated. Subsequently, the promoter activity was measured. The luciferase activity of mock-infected or unstimulated but empty vector-transfected cells was taken as unity. (C) A schedule of signaling cascades activated by IAV RNA is depicted (adapted from [ 10 , 55 , 56 ]). (D and E) HEK293 cells were transfected with the TopFlash reporter gene construct together with β-catenin and plasmids coding for the indicated proteins. At 30 h post transfection, the promoter activity was measured (D) . The luciferase activity of β-catenin and empty vector-transfected cells was arbitrarily taken as unity. Overexpression of the recombinant proteins was analyzed by Western blotting. The β-actin immunoblots always served as loading controls (E) . (F) A549 cells transfected with the TopFlash reporter plasmid together with β-catenin were stimulated with 5 mM of BAY inhibitor or DMSO as control. At 16 h post stimulation, cells were transfected with 500 ng cellular or viral RNA for an additional 8 h and the promoter activity was measured. The luciferase activity of cells stimulated with cellular RNA was always taken as unity. (G and H) Vero cells were transfected with reporter gene plasmids containing either the IRF3 responsive elements of the IFN-β enhanceosome (G) or the ISRE motif (H) along with plasmids coding for proteins indicated in column legends. The promoter activity was measured 30 h post transfection. Luciferase activity of cells transfected with any vector control was arbitrarily taken as unity.

    Techniques Used: Infection, Activation Assay, Transfection, Construct, Plasmid Preparation, Activity Assay, Luciferase, Over Expression, Recombinant, Western Blot

    7) Product Images from "Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses"

    Article Title: Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.109900

    Toxicity evaluation of the Cb AEs in Vero cells, A549 cells and ICR mice. (A, B) Cytotoxicity of Cb AEs to Vero cells (A) or A549 cells (B) was measured by MTT assays. (C-D) Toxicity assay of Cb AEs in ICR mice. (C) Mortality rate of acute intravenous (i.v.) administration of the Cb AEs in ICR mice (n=4 per group). (D) Body weight during the 7-day monitoring period in ICR mice subjected to acute intranasal (i.n.) administration of the Cb AEs (n=4 per group).
    Figure Legend Snippet: Toxicity evaluation of the Cb AEs in Vero cells, A549 cells and ICR mice. (A, B) Cytotoxicity of Cb AEs to Vero cells (A) or A549 cells (B) was measured by MTT assays. (C-D) Toxicity assay of Cb AEs in ICR mice. (C) Mortality rate of acute intravenous (i.v.) administration of the Cb AEs in ICR mice (n=4 per group). (D) Body weight during the 7-day monitoring period in ICR mice subjected to acute intranasal (i.n.) administration of the Cb AEs (n=4 per group).

    Techniques Used: Mouse Assay, MTT Assay

    8) Product Images from "RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma"

    Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24079

    AUF1 interacts with the coding region of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-ORF. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-ORF and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) EGFP protein expression in the treated Vero cells. n = 3. ** P
    Figure Legend Snippet: AUF1 interacts with the coding region of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-ORF. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-ORF and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) EGFP protein expression in the treated Vero cells. n = 3. ** P

    Techniques Used: Expressing, Transfection, Fluorescence, Microscopy

    AUF1 interacts with the 3′UTR of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-3′UTR. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-3′UTR and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) The expression of EGFP protein in the treated Vero cells. n = 3. ** P
    Figure Legend Snippet: AUF1 interacts with the 3′UTR of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-3′UTR. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-3′UTR and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) The expression of EGFP protein in the treated Vero cells. n = 3. ** P

    Techniques Used: Expressing, Transfection, Fluorescence, Microscopy

    9) Product Images from "Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies"

    Article Title: Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126127

    NS80 did not colocalize with outer-capsid proteins VP5 and VP7 in transfected or infected cells. (A, B, C) IF assay of NS80 and outer-capsid proteins in co-transfected cells. Vero cells were transfected with pCI-neo (A), pCI-neo-VP5 or pCI-neo-VP7 (C), or co-transfected with plasmids pCI-neo-NS80 and pCI-neo-VP5 or pCI-neo-VP7 (B) separately. (D, E) IF assay of NS80 and outer-capsid proteins in infected cells. Mock infected (D) or GCRV infected FHM cells (E) were fixed. After fixation, all cells were stained with mouse polyclonal antibodies against VP5 or VP7 (A-E), and with rabbit polyclonal antibody against NS80 (A, B, D and E). Cell nuclei were counterstained with Hoechst. Scale bars, 10 μm.
    Figure Legend Snippet: NS80 did not colocalize with outer-capsid proteins VP5 and VP7 in transfected or infected cells. (A, B, C) IF assay of NS80 and outer-capsid proteins in co-transfected cells. Vero cells were transfected with pCI-neo (A), pCI-neo-VP5 or pCI-neo-VP7 (C), or co-transfected with plasmids pCI-neo-NS80 and pCI-neo-VP5 or pCI-neo-VP7 (B) separately. (D, E) IF assay of NS80 and outer-capsid proteins in infected cells. Mock infected (D) or GCRV infected FHM cells (E) were fixed. After fixation, all cells were stained with mouse polyclonal antibodies against VP5 or VP7 (A-E), and with rabbit polyclonal antibody against NS80 (A, B, D and E). Cell nuclei were counterstained with Hoechst. Scale bars, 10 μm.

    Techniques Used: Transfection, Infection, Staining

    Interactions of NS80 with inner-capsid proteins (VP1-VP4 and VP6) in transfected cells. (A, B, C) IF assay of NS80 and inner-capsid proteins. Vero cells were transfected with pCI-neo (A), pCI-neo-VP1, pCI-neo-VP2, pCI-neo-VP3 or pCI-neo-VP4 plasmids (B), or co-transfected with plasmids pCI-neo-NS80 and pCI-neo-VP1, pCI-neo-VP2, pCI-neo-VP3 or pCI-neo-VP4 (C) separately. After fixation, cells were stained with mouse polyclonal antibodies against VP1, VP2, VP3 or VP4 (B), and with rabbit polyclonal antibody against NS80 (A and C) respectively. Cell nuclei were counterstained with Hoechst. Scale bars, 10 μm. (D-H) Co-IP assays of NS80 and inner-capsid proteins. HEK 293T cells were co-transfected with plasmids expressing NS80 and VP1 (E), VP2 (F), VP3 (G), VP4 (H) or VP6 respectively. Cells were lysed and IP assays were performed with polyclonal antibodies against NS80 (IP-NS80, D), VP1 (IP-VP1, E), VP2 (IP-VP2, F), VP3 (IP-VP3, G), VP4 (IP-VP4, H) or negative control serum (IP-IgG) respectively. IP samples were further analyzed by WB.
    Figure Legend Snippet: Interactions of NS80 with inner-capsid proteins (VP1-VP4 and VP6) in transfected cells. (A, B, C) IF assay of NS80 and inner-capsid proteins. Vero cells were transfected with pCI-neo (A), pCI-neo-VP1, pCI-neo-VP2, pCI-neo-VP3 or pCI-neo-VP4 plasmids (B), or co-transfected with plasmids pCI-neo-NS80 and pCI-neo-VP1, pCI-neo-VP2, pCI-neo-VP3 or pCI-neo-VP4 (C) separately. After fixation, cells were stained with mouse polyclonal antibodies against VP1, VP2, VP3 or VP4 (B), and with rabbit polyclonal antibody against NS80 (A and C) respectively. Cell nuclei were counterstained with Hoechst. Scale bars, 10 μm. (D-H) Co-IP assays of NS80 and inner-capsid proteins. HEK 293T cells were co-transfected with plasmids expressing NS80 and VP1 (E), VP2 (F), VP3 (G), VP4 (H) or VP6 respectively. Cells were lysed and IP assays were performed with polyclonal antibodies against NS80 (IP-NS80, D), VP1 (IP-VP1, E), VP2 (IP-VP2, F), VP3 (IP-VP3, G), VP4 (IP-VP4, H) or negative control serum (IP-IgG) respectively. IP samples were further analyzed by WB.

    Techniques Used: Transfection, Staining, Co-Immunoprecipitation Assay, Expressing, Negative Control, Western Blot

    10) Product Images from "Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis"

    Article Title: Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02316-14

    Cytotoxicity of wild-type and mutant TcdB. Vero cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and LDH activity in supernatants was
    Figure Legend Snippet: Cytotoxicity of wild-type and mutant TcdB. Vero cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and LDH activity in supernatants was

    Techniques Used: Mutagenesis, Activity Assay

    11) Product Images from "Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells"

    Article Title: Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells

    Journal: Infection and Immunity

    doi:

    Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.
    Figure Legend Snippet: Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.

    Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Infection

    Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.
    Figure Legend Snippet: Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Staining, Immunofluorescence

    12) Product Images from "Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan"

    Article Title: Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.257204

    Generation of PVR1 / PVR2 double-knockout (DKO) cell lines. ( a ) Diagram for construction of PVR1 / PVR2 double-deficient cell lines. In the first-round experiment, co-transfection of parental Vero cells with two plasmids expressing the sgRNA crV4 (targeting to PVR1 ) or crV3 (targeting to PVR2 ) gave only PVR1 single-knockout (SKO) cell lines as shown (Supplementary Fig. 2). One of the purified cell clones named Vero Δ PVR1 -1 was subjected to a second-round experiment, in which the cells were transfected with the crV3-expressing plasmid, and two PVR1/2 DKO cell lines named Vero Δ PVR1/2 -1 and Vero Δ PVR1/2 -2, respectively, were obtained (see also Supplementary Fig. 2). In another experiment, transfection of Vero cells with the plasmid expressing crV2 (targeting to PVR1 ) gave a PVR1 SKO cell line named Vero Δ PVR1 -2, and transfection of Vero Δ PVR1 -2 cells with the crV3-expressing plasmid gave four PVR1/2 DKO cell lines: Vero Δ PVR1/2 -3, Vero Δ PVR1/2 -4, Vero Δ PVR1/2 -5, and Vero Δ PVR1/2 -6 (Supplementary Fig. 2). For construction of PVR2 SKO cells, Vero JCRB9013 cells were transfected with the crV3-expressing plasmid, and two mutant cell lines, Vero Δ PVR2 -1 and Vero Δ PVR2 -2, were obtained (see also Supplementary Fig. 2). ( b ) Fifteen-microgram aliquots of protein samples prepared from the Vero cell line (Vero), two PVR1 SKO cell lines (Δ PVR1 -1 and Δ PVR1 -2), six PVR1 / PVR2 DKO cell lines (Δ PVR1/2 -1, Δ PVR1/2 -2, Δ PVR1/2 -3, Δ PVR1/2 -4, Δ PVR1/2 -5, and Δ PVR1/2 -6), and two PVR2 SKO cell lines (Δ PVR2 -1 and Δ PVR2 -2) were subjected to western blot analysis using the anti-PVR/CD155 rabbit monoclonal antibody D3G7H (upper panel). Loading control GAPDH was detected with the anti-GAPDH rabbit monoclonal antibody 14C1 (lower panel). ( c ) After removal of N-linked glycans, protein samples equivalent to 5 μg of cell lysate were subjected to western blot analysis. PNGase F-untreated controls of Vero and Δ PVR2 -1 cell-derived samples are shown in the leftmost and rightmost lanes, respectively, in the panel.
    Figure Legend Snippet: Generation of PVR1 / PVR2 double-knockout (DKO) cell lines. ( a ) Diagram for construction of PVR1 / PVR2 double-deficient cell lines. In the first-round experiment, co-transfection of parental Vero cells with two plasmids expressing the sgRNA crV4 (targeting to PVR1 ) or crV3 (targeting to PVR2 ) gave only PVR1 single-knockout (SKO) cell lines as shown (Supplementary Fig. 2). One of the purified cell clones named Vero Δ PVR1 -1 was subjected to a second-round experiment, in which the cells were transfected with the crV3-expressing plasmid, and two PVR1/2 DKO cell lines named Vero Δ PVR1/2 -1 and Vero Δ PVR1/2 -2, respectively, were obtained (see also Supplementary Fig. 2). In another experiment, transfection of Vero cells with the plasmid expressing crV2 (targeting to PVR1 ) gave a PVR1 SKO cell line named Vero Δ PVR1 -2, and transfection of Vero Δ PVR1 -2 cells with the crV3-expressing plasmid gave four PVR1/2 DKO cell lines: Vero Δ PVR1/2 -3, Vero Δ PVR1/2 -4, Vero Δ PVR1/2 -5, and Vero Δ PVR1/2 -6 (Supplementary Fig. 2). For construction of PVR2 SKO cells, Vero JCRB9013 cells were transfected with the crV3-expressing plasmid, and two mutant cell lines, Vero Δ PVR2 -1 and Vero Δ PVR2 -2, were obtained (see also Supplementary Fig. 2). ( b ) Fifteen-microgram aliquots of protein samples prepared from the Vero cell line (Vero), two PVR1 SKO cell lines (Δ PVR1 -1 and Δ PVR1 -2), six PVR1 / PVR2 DKO cell lines (Δ PVR1/2 -1, Δ PVR1/2 -2, Δ PVR1/2 -3, Δ PVR1/2 -4, Δ PVR1/2 -5, and Δ PVR1/2 -6), and two PVR2 SKO cell lines (Δ PVR2 -1 and Δ PVR2 -2) were subjected to western blot analysis using the anti-PVR/CD155 rabbit monoclonal antibody D3G7H (upper panel). Loading control GAPDH was detected with the anti-GAPDH rabbit monoclonal antibody 14C1 (lower panel). ( c ) After removal of N-linked glycans, protein samples equivalent to 5 μg of cell lysate were subjected to western blot analysis. PNGase F-untreated controls of Vero and Δ PVR2 -1 cell-derived samples are shown in the leftmost and rightmost lanes, respectively, in the panel.

    Techniques Used: Double Knockout, Cotransfection, Expressing, Knock-Out, Purification, Clone Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Derivative Assay

    13) Product Images from "Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus"

    Article Title: Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1608383113

    Cell surface expression and fusion-supporting ability of WT and mutant HN proteins. ( A ) Expression of WT (pink) and mutant (green) HN proteins on transfected HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells was examined by flow cytometry. Mutants have an alanine substituted for the residue interacting with the third sugar from the nonreducing terminal. The empty plasmid served as the control (black). ( B ) NCI-H358 and IMR-32 cells transfected with expression plasmids encoding the MuV-HN protein (WT or Y369A), MuV-F protein, and EGFP were observed for syncytia formation using fluorescent microscopy at 1 or 2 d after transfection. (Scale bar: 200 µm.) The data are representative of three independently performed experiments.
    Figure Legend Snippet: Cell surface expression and fusion-supporting ability of WT and mutant HN proteins. ( A ) Expression of WT (pink) and mutant (green) HN proteins on transfected HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells was examined by flow cytometry. Mutants have an alanine substituted for the residue interacting with the third sugar from the nonreducing terminal. The empty plasmid served as the control (black). ( B ) NCI-H358 and IMR-32 cells transfected with expression plasmids encoding the MuV-HN protein (WT or Y369A), MuV-F protein, and EGFP were observed for syncytia formation using fluorescent microscopy at 1 or 2 d after transfection. (Scale bar: 200 µm.) The data are representative of three independently performed experiments.

    Techniques Used: Expressing, Mutagenesis, Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Microscopy

    Involvement of the third sugar from the nonreducing terminal in the HN protein–receptor interaction. Experiments were performed for the HN protein of MuV ( A ) and PIV5, PIV2, and SeV ( B ). ( Left ) Aromatic residues involved in the interaction with the third sugar from the nonreducing terminal (Glc-3 of SL) as well as residues stacked with them in solved (MuV and PIV5) or model HN protein structures (PIV2 and SeV) are shown in magenta. SLs (Sia-1, Gal-2, and Glc-3) are shown in cyan. ( Right ) HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells transfected with expression plasmids encoding the HN protein (WT or mutant), F protein, and EGFP were observed for syncytia formation using fluorescence microscopy at 2 d posttransfection. (Scale bar: 200 μm.)
    Figure Legend Snippet: Involvement of the third sugar from the nonreducing terminal in the HN protein–receptor interaction. Experiments were performed for the HN protein of MuV ( A ) and PIV5, PIV2, and SeV ( B ). ( Left ) Aromatic residues involved in the interaction with the third sugar from the nonreducing terminal (Glc-3 of SL) as well as residues stacked with them in solved (MuV and PIV5) or model HN protein structures (PIV2 and SeV) are shown in magenta. SLs (Sia-1, Gal-2, and Glc-3) are shown in cyan. ( Right ) HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells transfected with expression plasmids encoding the HN protein (WT or mutant), F protein, and EGFP were observed for syncytia formation using fluorescence microscopy at 2 d posttransfection. (Scale bar: 200 μm.)

    Techniques Used: Gas Chromatography, Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy

    14) Product Images from "Capsid Protein of Eastern Equine Encephalitis Virus Inhibits Host Cell Gene Expression ▿"

    Article Title: Capsid Protein of Eastern Equine Encephalitis Virus Inhibits Host Cell Gene Expression ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02075-06

    EEEV capsid is a general inhibitor of gene expression. (A and B) Vero cells were cotransfected with the ISG54 promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV capsid or Nipah virus V protein. At 24 hours posttransfection, the cells were mock treated or treated with 1,000 U human IFN-β/ml for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (A) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (B) Relative CAT activity upon normalization to the luciferase control. (C and D) 293T cells were cotransfected with the IFN-β promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV nsP2, EEEV capsid, Ebola virus VP35, or Nipah virus W. At 24 hours posttransfection, the cells were mock infected or infected with Sendai virus (SeV) for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (C) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (D) CAT activity upon normalization to the luciferase control. The results are representative examples from a set of at least three separate experiments. (E) 293T cells were cotransfected with a luciferase reporter plasmid and either empty vector or a plasmid expressing EEEV capsid. At 24 h posttransfection, the cells were harvested and assayed for luciferase activity. The data represent the means ± standard errors among samples from three separately transfected wells. The experiment was repeated at least twice, with consistent results. (F) An experiment was performed as described for panel E, except that a plasmid expressing GFP was used and cells were examined by microscopy for GFP expression. The experiment was repeated at least twice, with consistent results.
    Figure Legend Snippet: EEEV capsid is a general inhibitor of gene expression. (A and B) Vero cells were cotransfected with the ISG54 promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV capsid or Nipah virus V protein. At 24 hours posttransfection, the cells were mock treated or treated with 1,000 U human IFN-β/ml for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (A) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (B) Relative CAT activity upon normalization to the luciferase control. (C and D) 293T cells were cotransfected with the IFN-β promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV nsP2, EEEV capsid, Ebola virus VP35, or Nipah virus W. At 24 hours posttransfection, the cells were mock infected or infected with Sendai virus (SeV) for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (C) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (D) CAT activity upon normalization to the luciferase control. The results are representative examples from a set of at least three separate experiments. (E) 293T cells were cotransfected with a luciferase reporter plasmid and either empty vector or a plasmid expressing EEEV capsid. At 24 h posttransfection, the cells were harvested and assayed for luciferase activity. The data represent the means ± standard errors among samples from three separately transfected wells. The experiment was repeated at least twice, with consistent results. (F) An experiment was performed as described for panel E, except that a plasmid expressing GFP was used and cells were examined by microscopy for GFP expression. The experiment was repeated at least twice, with consistent results.

    Techniques Used: Expressing, Plasmid Preparation, Luciferase, Activity Assay, Infection, Transfection, Microscopy

    15) Product Images from "Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Inhibits Beta Interferon Production by Deubiquitinating TRAF3"

    Article Title: Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Inhibits Beta Interferon Production by Deubiquitinating TRAF3

    Journal: Journal of Virology

    doi: 10.1128/JVI.01211-13

    The UL36USP C40A mutation reduces the replication of recombinant virus in HEK293T cells. (A) Schematic diagram of construction of the C40A HSV-1 BAC. Cys40 of UL36 was substituted for by Ala in the HSV-1 genome. Confluent Vero cells were infected with
    Figure Legend Snippet: The UL36USP C40A mutation reduces the replication of recombinant virus in HEK293T cells. (A) Schematic diagram of construction of the C40A HSV-1 BAC. Cys40 of UL36 was substituted for by Ala in the HSV-1 genome. Confluent Vero cells were infected with

    Techniques Used: Mutagenesis, Recombinant, BAC Assay, Infection

    16) Product Images from "Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes"

    Article Title: Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006958

    At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.
    Figure Legend Snippet: At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.

    Techniques Used: Expressing, Mutagenesis, Infection, Staining, Microscopy, Transfection, Fluorescence, Immunoprecipitation, Western Blot

    At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.
    Figure Legend Snippet: At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.

    Techniques Used: Transfection, In Vitro, Staining, Fluorescence, Microscopy, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

    The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.
    Figure Legend Snippet: The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.

    Techniques Used: Transfection, In Vitro, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

    A single FGDF motif is sufficient for the transmission of chikungunya virus by Aedes aegypti mosquitoes. (A) Schematic experimental set-up. Female Ae . aegypti mosquitoes were infected through an infectious bloodmeal containing 2.8 × 10 5 TCID 50 /ml of CHIK IC , CHIK IC -FG N , CHIK IC -FG C. At 7 days post infection (7dpi) the infection and transmission rates were determined by infectivity assay on (B) Vero and (C) Aag2 cells. Bars represent cumulative numbers from three independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) Ratio between the number of positive bodies or salivas in Vero compared to Aag2 cells. Statistics were performed by Kruskal-Wallis test with Dunn’s post-hoc test (α = 0.05). (E) TCID 50 /ml in the bodies of Ae . aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Aag2 cells. Statistics was performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data (α = 0.05).
    Figure Legend Snippet: A single FGDF motif is sufficient for the transmission of chikungunya virus by Aedes aegypti mosquitoes. (A) Schematic experimental set-up. Female Ae . aegypti mosquitoes were infected through an infectious bloodmeal containing 2.8 × 10 5 TCID 50 /ml of CHIK IC , CHIK IC -FG N , CHIK IC -FG C. At 7 days post infection (7dpi) the infection and transmission rates were determined by infectivity assay on (B) Vero and (C) Aag2 cells. Bars represent cumulative numbers from three independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) Ratio between the number of positive bodies or salivas in Vero compared to Aag2 cells. Statistics were performed by Kruskal-Wallis test with Dunn’s post-hoc test (α = 0.05). (E) TCID 50 /ml in the bodies of Ae . aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Aag2 cells. Statistics was performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data (α = 0.05).

    Techniques Used: Transmission Assay, Infection, End-point Dilution Assay, Transformation Assay

    17) Product Images from "Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress"

    Article Title: Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress

    Journal: Virology

    doi: 10.1016/j.virol.2005.11.053

    Regulation of U L 34-mediated lamin disruption by co-expression of U S 3. For all panels, Vero cells were transfected with plasmids expressing U L 34 and U S 3or kinase-dead U S 3 for 72 h. (A–Q) Digital confocal images showing the localization of U L 34, U S 3 or kinase-dead U S 3 and lamin A/C or lamin B in Vero cells transfected with plasmids expressing U L 34 and U S 3 or kinase-dead U S 3. All cells were transfected with U L 34-expressing plasmid. The identity of the US3-expressing plasmid is indicated to the left of each row. The protein being detected is indicated above each column of panels. Green: lamins, red: U L 34, blue: U S 3 (Q and R) Cells transfected with U L 34 or U S 3 alone or together were analyzed by flow cytometry. (Q) U L 34 staining profile for untransfected cells (red) or cells transfected with U L 34 (blue) or U L 34 and U S 3 (green). (R) U S 3 staining profile for untransfected cells (red) cells transfected with U S 3 (blue) or U S 3 and U L 34 (green).
    Figure Legend Snippet: Regulation of U L 34-mediated lamin disruption by co-expression of U S 3. For all panels, Vero cells were transfected with plasmids expressing U L 34 and U S 3or kinase-dead U S 3 for 72 h. (A–Q) Digital confocal images showing the localization of U L 34, U S 3 or kinase-dead U S 3 and lamin A/C or lamin B in Vero cells transfected with plasmids expressing U L 34 and U S 3 or kinase-dead U S 3. All cells were transfected with U L 34-expressing plasmid. The identity of the US3-expressing plasmid is indicated to the left of each row. The protein being detected is indicated above each column of panels. Green: lamins, red: U L 34, blue: U S 3 (Q and R) Cells transfected with U L 34 or U S 3 alone or together were analyzed by flow cytometry. (Q) U L 34 staining profile for untransfected cells (red) or cells transfected with U L 34 (blue) or U L 34 and U S 3 (green). (R) U S 3 staining profile for untransfected cells (red) cells transfected with U S 3 (blue) or U S 3 and U L 34 (green).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Staining

    Disruptions in lamin localization are not accompanied by loss of lamin protein or cleavage of PARP. (A) Lamin A/C protein levels in Vero cells mock-infected (red), or infected with HSV-1(F) (blue) or vRR1202 (green), as analyzed by flow cytometry. The left histogram shows the results of detection with no primary antibody; the right graph shows the results of detection with both primary and secondary antibodies. (B) Lamin B protein levels in Vero cells mock-infected (lane 1), or infected with HSV-1(F) (lane 2) or vRR1202 (lane 3), as analyzed by western blot. Total cellular protein was extracted as described in Materials and methods. Coomassie staining was used to equilibrate loading (not shown). These experiments were done three times. Representative images are shown. Lane 3 is from the same blot and the same exposure as lanes 1 and 2, but was not adjacent to these lanes. (C and D) Digital confocal merged images showing the presence of cleaved PARP positive cells in Vero cells infected with vRR1202 or vR1204 at an MOI of 5 for 24 h. This experiment was done five times. A representative experiment is shown. The infecting virus is indicated on each panel. Green: lamin A/C; blue: cleaved PARP. The arrows point to cells that have perforations in the lamina, but do not stain positive for cleaved PARP. (E) Vero cells were transfected with a plasmid expressing either wild type lamin B-FLAG or non-cleavable lamin B-FLAG, then mock-infected or infected with vRR1202. The identity of the transfecting plasmid is indicated below each column. The state of infection is indicated to the right of each row.
    Figure Legend Snippet: Disruptions in lamin localization are not accompanied by loss of lamin protein or cleavage of PARP. (A) Lamin A/C protein levels in Vero cells mock-infected (red), or infected with HSV-1(F) (blue) or vRR1202 (green), as analyzed by flow cytometry. The left histogram shows the results of detection with no primary antibody; the right graph shows the results of detection with both primary and secondary antibodies. (B) Lamin B protein levels in Vero cells mock-infected (lane 1), or infected with HSV-1(F) (lane 2) or vRR1202 (lane 3), as analyzed by western blot. Total cellular protein was extracted as described in Materials and methods. Coomassie staining was used to equilibrate loading (not shown). These experiments were done three times. Representative images are shown. Lane 3 is from the same blot and the same exposure as lanes 1 and 2, but was not adjacent to these lanes. (C and D) Digital confocal merged images showing the presence of cleaved PARP positive cells in Vero cells infected with vRR1202 or vR1204 at an MOI of 5 for 24 h. This experiment was done five times. A representative experiment is shown. The infecting virus is indicated on each panel. Green: lamin A/C; blue: cleaved PARP. The arrows point to cells that have perforations in the lamina, but do not stain positive for cleaved PARP. (E) Vero cells were transfected with a plasmid expressing either wild type lamin B-FLAG or non-cleavable lamin B-FLAG, then mock-infected or infected with vRR1202. The identity of the transfecting plasmid is indicated below each column. The state of infection is indicated to the right of each row.

    Techniques Used: Infection, Flow Cytometry, Cytometry, Western Blot, Staining, Transfection, Plasmid Preparation, Expressing

    Disruption of Lamin A/C and lamin B localization by expression of transfected U L 34 or CD4 genes. Shown are digital confocal images showing the localization of U L 34, CD4 and lamin A/C or lamin B in Vero cells expressing either the U L 34 or CD4 protein for various times. Green: lamins, red: U L 34, blue: CD4. For all panels, Vero cells were transfected with plasmids expressing either U L 34 or CD4 for 24, 48, or 72 h. Time after transfection is indicated to the left of each row of panels. The protein being detected is indicated above each column of panels.
    Figure Legend Snippet: Disruption of Lamin A/C and lamin B localization by expression of transfected U L 34 or CD4 genes. Shown are digital confocal images showing the localization of U L 34, CD4 and lamin A/C or lamin B in Vero cells expressing either the U L 34 or CD4 protein for various times. Green: lamins, red: U L 34, blue: CD4. For all panels, Vero cells were transfected with plasmids expressing either U L 34 or CD4 for 24, 48, or 72 h. Time after transfection is indicated to the left of each row of panels. The protein being detected is indicated above each column of panels.

    Techniques Used: Expressing, Transfection

    Disruption of lamin A/C and lamin B localization by expression of a transfected U S 3 gene. Shown are digital merged images showing the localization of wild type U S 3, mutant U S 3, beta-galactosidase and lamin A/C or lamin B in Vero cells transfected with plasmids expressing U S 3 or beta-galactosidase protein. Green: lamins, blue: U S 3 or beta-galactosidase. For all panels A–L, Vero cells were transfected with a plasmid expressing wild-type or kinase dead U S 3 for 24, 48 and 72 h. For panels M and N, only the 72 h time point is shown. Time after transfection is indicated to the left of each row of panels. The proteins being detected are indicated above each column of panels.
    Figure Legend Snippet: Disruption of lamin A/C and lamin B localization by expression of a transfected U S 3 gene. Shown are digital merged images showing the localization of wild type U S 3, mutant U S 3, beta-galactosidase and lamin A/C or lamin B in Vero cells transfected with plasmids expressing U S 3 or beta-galactosidase protein. Green: lamins, blue: U S 3 or beta-galactosidase. For all panels A–L, Vero cells were transfected with a plasmid expressing wild-type or kinase dead U S 3 for 24, 48 and 72 h. For panels M and N, only the 72 h time point is shown. Time after transfection is indicated to the left of each row of panels. The proteins being detected are indicated above each column of panels.

    Techniques Used: Expressing, Transfection, Mutagenesis, Plasmid Preparation

    18) Product Images from "MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism"

    Article Title: MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism

    Journal: Molecular Therapy

    doi: 10.1038/mt.2011.55

    miR7 regulates spreading, cytotoxicity, protein expression, and progeny virus production of MV-EGFP miR7 . ( a ) Vero cells were seeded in 6-well plates at a density of 6 × 10 5 cells/well. After attachment, cells were transfected with 0 or 20 nmol/l
    Figure Legend Snippet: miR7 regulates spreading, cytotoxicity, protein expression, and progeny virus production of MV-EGFP miR7 . ( a ) Vero cells were seeded in 6-well plates at a density of 6 × 10 5 cells/well. After attachment, cells were transfected with 0 or 20 nmol/l

    Techniques Used: Expressing, Transfection

    Syncytia formation and expression of the measles virus (MV) F gene containing microRNA-target sites are repressed by the presence of miR7 . ( a ) 2 × 10 5 Vero cells/well were seeded in 12-well plates and after attachment co-transfected with 0 or
    Figure Legend Snippet: Syncytia formation and expression of the measles virus (MV) F gene containing microRNA-target sites are repressed by the presence of miR7 . ( a ) 2 × 10 5 Vero cells/well were seeded in 12-well plates and after attachment co-transfected with 0 or

    Techniques Used: Expressing, Transfection

    19) Product Images from "De Novo Generation and Characterization of New Zika Virus Isolate Using Sequence Data from a Microcephaly Case"

    Article Title: De Novo Generation and Characterization of New Zika Virus Isolate Using Sequence Data from a Microcephaly Case

    Journal: mSphere

    doi: 10.1128/mSphereDirect.00190-17

    De novo generation of infectious ZIKV Natal by CPER. (A) Schematic representation of CPER assembly with seven synthetic DNA fragments covering the entire genome of ZIKV Natal with ~22-nt overlapping ends. The fragments were mixed with the UTR linker, which contained the CMV promoter followed by the first 22 nt of ZIKV Natal and, at the other end, the last 22 nt of ZIKV Natal , an HDVr site, and a poly(A) tail (pA). After CPER, the products were transfected into Vero cells. (B) On the posttransfection days indicated, RNA was isolated from Vero cell supernatants and a cell lysate and subjected to RT-PCR with fragment 5-specific primers (RT-PCR). To demonstrate that the bands were not due to contaminating DNA, a control (RT minus) PCR was performed in parallel (PCR). (C) Vero cells were infected with ZIKV Natal (culture supernatant from day 10 posttransfection), and mock-infected cells were used as controls. At 3 days postinfection, Vero cells were analyzed by immunofluorescent-antibody staining with anti-NS1 (4G4) and anti-dsRNA (3G1) antibodies (green) and DAPI counterstain (blue). (D) Plaque morphology following infection of Vero cells with ZIKV Natal and ZIKV MR766 . Vero cells were fixed and stained with crystal violet on the postinfection days indicated. (E) Growth kinetics of ZIKV Natal and ZIKV MR766 in the cell lines indicated. Infection was performed at an MOI of 0.1, unless otherwise indicated. Virus titers in the culture supernatants were determined by plaque assays on Vero cells. The data and standard errors (SE) shown are from six independent experiments with Vero cells, A549 cells, and WT MEFs (MOI of 1) and three independent experiments with WT MEFs (MOI of 0.1), IFNAR −/− MEFs, and C6/36 cells. The horizontal line represents the limit of detection (50 PFU/ml).
    Figure Legend Snippet: De novo generation of infectious ZIKV Natal by CPER. (A) Schematic representation of CPER assembly with seven synthetic DNA fragments covering the entire genome of ZIKV Natal with ~22-nt overlapping ends. The fragments were mixed with the UTR linker, which contained the CMV promoter followed by the first 22 nt of ZIKV Natal and, at the other end, the last 22 nt of ZIKV Natal , an HDVr site, and a poly(A) tail (pA). After CPER, the products were transfected into Vero cells. (B) On the posttransfection days indicated, RNA was isolated from Vero cell supernatants and a cell lysate and subjected to RT-PCR with fragment 5-specific primers (RT-PCR). To demonstrate that the bands were not due to contaminating DNA, a control (RT minus) PCR was performed in parallel (PCR). (C) Vero cells were infected with ZIKV Natal (culture supernatant from day 10 posttransfection), and mock-infected cells were used as controls. At 3 days postinfection, Vero cells were analyzed by immunofluorescent-antibody staining with anti-NS1 (4G4) and anti-dsRNA (3G1) antibodies (green) and DAPI counterstain (blue). (D) Plaque morphology following infection of Vero cells with ZIKV Natal and ZIKV MR766 . Vero cells were fixed and stained with crystal violet on the postinfection days indicated. (E) Growth kinetics of ZIKV Natal and ZIKV MR766 in the cell lines indicated. Infection was performed at an MOI of 0.1, unless otherwise indicated. Virus titers in the culture supernatants were determined by plaque assays on Vero cells. The data and standard errors (SE) shown are from six independent experiments with Vero cells, A549 cells, and WT MEFs (MOI of 1) and three independent experiments with WT MEFs (MOI of 0.1), IFNAR −/− MEFs, and C6/36 cells. The horizontal line represents the limit of detection (50 PFU/ml).

    Techniques Used: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Infection, Staining

    20) Product Images from "Etlingera elatior Extract promotes cell death in B16 melanoma cells via down-regulation of ERK and Akt signaling pathways"

    Article Title: Etlingera elatior Extract promotes cell death in B16 melanoma cells via down-regulation of ERK and Akt signaling pathways

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-017-1921-y

    EE extract inhibited cell viability in B16 cells. The cytotoxic effect of EE extract was performed using the MTT assay. ( a ) B16 and ( b ) Vero cells were treated with various concentrations of EE extract at different time points for 24 h. Results are mean values ± SD of three independent experiments ( n = 3). * p
    Figure Legend Snippet: EE extract inhibited cell viability in B16 cells. The cytotoxic effect of EE extract was performed using the MTT assay. ( a ) B16 and ( b ) Vero cells were treated with various concentrations of EE extract at different time points for 24 h. Results are mean values ± SD of three independent experiments ( n = 3). * p

    Techniques Used: MTT Assay

    21) Product Images from "Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes"

    Article Title: Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

    Journal: Journal of Virology

    doi: 10.1128/JVI.00057-15

    Fusion pH as detected by luciferase reporter gene assay. HA-expressing Vero cells transfected with T7-luciferase plasmid were treated with TPCK-trypsin (5 μg/ml), followed by soybean trypsin inhibitor (20 μg/ml), and then overlaid with
    Figure Legend Snippet: Fusion pH as detected by luciferase reporter gene assay. HA-expressing Vero cells transfected with T7-luciferase plasmid were treated with TPCK-trypsin (5 μg/ml), followed by soybean trypsin inhibitor (20 μg/ml), and then overlaid with

    Techniques Used: Luciferase, Reporter Gene Assay, Expressing, Transfection, Plasmid Preparation

    22) Product Images from "Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿ †"

    Article Title: Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00949-10

    Mutation of a conserved proline in nsP2 abolishes the inhibitory effect of CHIKV and SINV replicons on JAK-STAT signaling. (A) Schematic representation of the CHIKrep-pac2AEGFP and SINrepLuc replicons. nsP2 mutations P718S and P726S are indicated with asterisks; pac, puromycin acetyltransferase. (B) Partial amino acid alignment of alphavirus nsP2s. RRV, Ross River virus; VEEV, Venezuelan equine encephalitis virus. The conserved proline and amino acid numbers within nsP2 proteins are indicated. (C) pSTAT1 nuclear translocation upon IFN-β induction in SINrepGFP (wild type and mutant nsP2-P726S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody. Open arrowheads indicate replicon-positive cells lacking nuclear pSTAT1; solid arrowheads indicate replicon-positive cells with nuclear pSTAT1. (D) Nuclear translocation of phospho-STAT1 upon IFN-β induction in CHIKrep-pac2AEGFP (wild type and mutant nsP2-P718S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody.
    Figure Legend Snippet: Mutation of a conserved proline in nsP2 abolishes the inhibitory effect of CHIKV and SINV replicons on JAK-STAT signaling. (A) Schematic representation of the CHIKrep-pac2AEGFP and SINrepLuc replicons. nsP2 mutations P718S and P726S are indicated with asterisks; pac, puromycin acetyltransferase. (B) Partial amino acid alignment of alphavirus nsP2s. RRV, Ross River virus; VEEV, Venezuelan equine encephalitis virus. The conserved proline and amino acid numbers within nsP2 proteins are indicated. (C) pSTAT1 nuclear translocation upon IFN-β induction in SINrepGFP (wild type and mutant nsP2-P726S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody. Open arrowheads indicate replicon-positive cells lacking nuclear pSTAT1; solid arrowheads indicate replicon-positive cells with nuclear pSTAT1. (D) Nuclear translocation of phospho-STAT1 upon IFN-β induction in CHIKrep-pac2AEGFP (wild type and mutant nsP2-P718S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody.

    Techniques Used: Mutagenesis, Translocation Assay, Transfection

    Inhibition of IFN-β-induced STAT1 nuclear translocation by individual CHIKV nsPs. (A) Schematic representation of the pCMV-nsP1, -2, -3, and -4 expression plasmids and the CHIKrep-mCherry replicon, expressing mCherry. CMV, cytomegalovirus immediate-early promoter; 2A, foot-and-mouth disease virus 2A autoprotease. The bacteriophage SP6 and CHIKV 26S promoters are indicated. (B) pSTAT1 nuclear translocation upon IFN-β induction in Vero cells transfected with pCMV-nsP1, -2, -3, or -4. Cells were immunostained with an anti-pSTAT1 antibody. (C) pSTAT1 nuclear translocation upon IFN-β induction in CHIKrep-mCherry-transfected Vero cells. Open arrowheads indicate cells positive for nsP1, -2, -3, or -4- or for the CHIKV replicon that lack nuclear pSTAT1; solid arrowheads indicate nsP1- to nsP4-positive cells with nuclear pSTAT1.
    Figure Legend Snippet: Inhibition of IFN-β-induced STAT1 nuclear translocation by individual CHIKV nsPs. (A) Schematic representation of the pCMV-nsP1, -2, -3, and -4 expression plasmids and the CHIKrep-mCherry replicon, expressing mCherry. CMV, cytomegalovirus immediate-early promoter; 2A, foot-and-mouth disease virus 2A autoprotease. The bacteriophage SP6 and CHIKV 26S promoters are indicated. (B) pSTAT1 nuclear translocation upon IFN-β induction in Vero cells transfected with pCMV-nsP1, -2, -3, or -4. Cells were immunostained with an anti-pSTAT1 antibody. (C) pSTAT1 nuclear translocation upon IFN-β induction in CHIKrep-mCherry-transfected Vero cells. Open arrowheads indicate cells positive for nsP1, -2, -3, or -4- or for the CHIKV replicon that lack nuclear pSTAT1; solid arrowheads indicate nsP1- to nsP4-positive cells with nuclear pSTAT1.

    Techniques Used: Inhibition, Translocation Assay, Expressing, Transfection

    Inhibition of type I/II IFN signaling and ISG induction by CHIKV infection. (A and B) Vero cells were transfected with a pRL-TK plasmid expressing Rluc and either a type I IFN-responsive (ISRE) or a type II IFN-responsive (GAS) Fluc reporter plasmid. At 24 h p.t., cells were infected with CHIKV at an MOI of 5 PFU/ml. At 4, 8, and 12 h p.i., cells were treated with IFN-α at 1,000 IU/ml (A) or with IFN-γ at 100 ng/ml (B) for 6 h; then they were assayed for Fluc and Rluc activities. Activities in mock-infected (uninfected) cells with/without IFN induction were also measured. Fluc values were divided by Rluc readings to compensate for virus-induced downregulation of transcription/translation and were expressed relative to values for mock-infected, IFN-treated samples. Average values from triplicate samples are shown. Error bars represent standard deviations. (C and D) Vero cells, either healthy or infected with CHIKV for 4, 8, or 12 h, were incubated with 1,000 IU of IFN-α (C) or 100 ng of IFN-γ (D) per ml for 10 h. Real-time RT-PCR values for the IFN-stimulated gene OAS2 were normalized to those for the housekeeping gene RPL13A. OAS2 mRNA transcription levels were expressed relative to those of mock-infected, IFN-treated samples. Average values from duplicate samples are shown. Error bars represent standard deviations.
    Figure Legend Snippet: Inhibition of type I/II IFN signaling and ISG induction by CHIKV infection. (A and B) Vero cells were transfected with a pRL-TK plasmid expressing Rluc and either a type I IFN-responsive (ISRE) or a type II IFN-responsive (GAS) Fluc reporter plasmid. At 24 h p.t., cells were infected with CHIKV at an MOI of 5 PFU/ml. At 4, 8, and 12 h p.i., cells were treated with IFN-α at 1,000 IU/ml (A) or with IFN-γ at 100 ng/ml (B) for 6 h; then they were assayed for Fluc and Rluc activities. Activities in mock-infected (uninfected) cells with/without IFN induction were also measured. Fluc values were divided by Rluc readings to compensate for virus-induced downregulation of transcription/translation and were expressed relative to values for mock-infected, IFN-treated samples. Average values from triplicate samples are shown. Error bars represent standard deviations. (C and D) Vero cells, either healthy or infected with CHIKV for 4, 8, or 12 h, were incubated with 1,000 IU of IFN-α (C) or 100 ng of IFN-γ (D) per ml for 10 h. Real-time RT-PCR values for the IFN-stimulated gene OAS2 were normalized to those for the housekeeping gene RPL13A. OAS2 mRNA transcription levels were expressed relative to those of mock-infected, IFN-treated samples. Average values from duplicate samples are shown. Error bars represent standard deviations.

    Techniques Used: Inhibition, Infection, Transfection, Plasmid Preparation, Expressing, Incubation, Quantitative RT-PCR

    A CHIKV replicon efficiently inhibits type I/II IFN-induced JAK-STAT signaling independently of host shutoff. (A) Schematic representation of CHIKrepEGFP, expressing EGFP. (B) pSTAT1 nuclear translocation in Vero cells upon induction with type I and type II IFNs. (C) A CHIKV replicon blocks pSTAT1 nuclear translocation upon type I/II IFN induction. Vero cells were immunostained with an anti-pSTAT1 antibody 24 h p.t. (D) CHIKV RNA replication, but not translational shutoff, blocks STAT1 nuclear translocation. Vero cells were transfected with CHIKrep-EGFP replicon RNA in the absence or presence of cycloheximide (Chx). Cells were induced for 30 min with IFN-β at 12 h p.t. and were stained with an anti-STAT1 antibody. Open arrowheads indicate CHIKV replicon-positive cells lacking nuclear STAT1.
    Figure Legend Snippet: A CHIKV replicon efficiently inhibits type I/II IFN-induced JAK-STAT signaling independently of host shutoff. (A) Schematic representation of CHIKrepEGFP, expressing EGFP. (B) pSTAT1 nuclear translocation in Vero cells upon induction with type I and type II IFNs. (C) A CHIKV replicon blocks pSTAT1 nuclear translocation upon type I/II IFN induction. Vero cells were immunostained with an anti-pSTAT1 antibody 24 h p.t. (D) CHIKV RNA replication, but not translational shutoff, blocks STAT1 nuclear translocation. Vero cells were transfected with CHIKrep-EGFP replicon RNA in the absence or presence of cycloheximide (Chx). Cells were induced for 30 min with IFN-β at 12 h p.t. and were stained with an anti-STAT1 antibody. Open arrowheads indicate CHIKV replicon-positive cells lacking nuclear STAT1.

    Techniques Used: Expressing, Translocation Assay, Transfection, Staining

    23) Product Images from "Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses"

    Article Title: Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.13.8004-8013.2005

    Expression of 2K-mutant NS4Bs. (A) Induction of ISRE-9-27-CAT reporter gene after treatment with IFN. Recombinant pCAGGS-HA plasmids containing the DEN-2 NS4B, Δ2K-NS4B, or KB-NS4B gene fragments were transfected in Vero cells along with reporter
    Figure Legend Snippet: Expression of 2K-mutant NS4Bs. (A) Induction of ISRE-9-27-CAT reporter gene after treatment with IFN. Recombinant pCAGGS-HA plasmids containing the DEN-2 NS4B, Δ2K-NS4B, or KB-NS4B gene fragments were transfected in Vero cells along with reporter

    Techniques Used: Expressing, Mutagenesis, Recombinant, Transfection

    Analysis of C-terminal deletions of DEN NS4B. (A) Induction of ISRE-9-27-CAT reporter gene after treatment with IFN. Recombinant pCAGGS-HA plasmids containing each of the DEN-2 NS4B C-terminal deletions were transfected in Vero cells together with the
    Figure Legend Snippet: Analysis of C-terminal deletions of DEN NS4B. (A) Induction of ISRE-9-27-CAT reporter gene after treatment with IFN. Recombinant pCAGGS-HA plasmids containing each of the DEN-2 NS4B C-terminal deletions were transfected in Vero cells together with the

    Techniques Used: Recombinant, Transfection

    Conserved NS4B function among flaviviruses. (A) An immunoblot shows levels of expression of DEN, WNV, and YFV NS4B in Vero cells transfected with the indicated plasmids. (B) Induction of ISRE-9-27-CAT after treatment with IFN-β. Vero cells were
    Figure Legend Snippet: Conserved NS4B function among flaviviruses. (A) An immunoblot shows levels of expression of DEN, WNV, and YFV NS4B in Vero cells transfected with the indicated plasmids. (B) Induction of ISRE-9-27-CAT after treatment with IFN-β. Vero cells were

    Techniques Used: Expressing, Transfection

    24) Product Images from "Virus-Induced Chaperone-Enriched (VICE) Domains Function as Nuclear Protein Quality Control Centers during HSV-1 Infection"

    Article Title: Virus-Induced Chaperone-Enriched (VICE) Domains Function as Nuclear Protein Quality Control Centers during HSV-1 Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000619

    Homotypic ubiquitin chains are not detected in VICE domains. Vero cells adhered to glass coverslips were transfected with 1 ug of pRK-HA-Ub constructs encoding wt, K63, K48 and K48R Ub (panel A) and K0 (monomeric), K6, K11, K27, K29 and K33 Ub (panel B). Following 16–18 hours of protein expression, transfected cells were infected with KOS at an MOI of 10 for 6 hours. At harvest, cells were prepared for IF analysis and labeled with mouse-anti-HA tag, rabbit-anti-ICP8 367 and rat-anti-Hsc70 antibodies. Images were taken at 63× magnification with 2× digital zoom. Merged images of green, red and far-red channels are shown.
    Figure Legend Snippet: Homotypic ubiquitin chains are not detected in VICE domains. Vero cells adhered to glass coverslips were transfected with 1 ug of pRK-HA-Ub constructs encoding wt, K63, K48 and K48R Ub (panel A) and K0 (monomeric), K6, K11, K27, K29 and K33 Ub (panel B). Following 16–18 hours of protein expression, transfected cells were infected with KOS at an MOI of 10 for 6 hours. At harvest, cells were prepared for IF analysis and labeled with mouse-anti-HA tag, rabbit-anti-ICP8 367 and rat-anti-Hsc70 antibodies. Images were taken at 63× magnification with 2× digital zoom. Merged images of green, red and far-red channels are shown.

    Techniques Used: Transfection, Construct, Expressing, Infection, Labeling

    FRAP analysis of Hsc70-GFP in VICE domains. Vero cells adhered to a coverslip dish were transfected with 1 ug of a plasmid encoding wt Hsc70-GFP. Approximately 16 hours post transfection, cells were infected with KOS at an MOI of 10. At 5 hours post infection, live infected cells were imaged on the LSM 510 Meta confocal microscope. At time 0, Hsc70-GFP in one VICE domain was photobleached using 100% argon laser power, and subsequent images collected at ∼2.5 s intervals. (A) Single images from the time series are shown; arrow indicates the VICE domain selected for photobleaching. (B) Fluorescence intensity in the bleached VICE domain (circles), a VICE domain that was not photobleached (squares) and an equivalently sized area of the nucleoplasm (triangles) is shown. Data are normalized to prebleach intensity and corrected for bleaching during monitoring. The experiment shown is representative of at least three independent experiments each involving analysis of numerous infected cells.
    Figure Legend Snippet: FRAP analysis of Hsc70-GFP in VICE domains. Vero cells adhered to a coverslip dish were transfected with 1 ug of a plasmid encoding wt Hsc70-GFP. Approximately 16 hours post transfection, cells were infected with KOS at an MOI of 10. At 5 hours post infection, live infected cells were imaged on the LSM 510 Meta confocal microscope. At time 0, Hsc70-GFP in one VICE domain was photobleached using 100% argon laser power, and subsequent images collected at ∼2.5 s intervals. (A) Single images from the time series are shown; arrow indicates the VICE domain selected for photobleaching. (B) Fluorescence intensity in the bleached VICE domain (circles), a VICE domain that was not photobleached (squares) and an equivalently sized area of the nucleoplasm (triangles) is shown. Data are normalized to prebleach intensity and corrected for bleaching during monitoring. The experiment shown is representative of at least three independent experiments each involving analysis of numerous infected cells.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Microscopy, Fluorescence

    A model misfolded protein is localized to VICE domains. Vero cells were transfected with 1 ug of plasmid DNA encoding the model misfolded protein GFP170* [2] . Following about 16 hours of protein expression, the cells were infected with KOS at an MOI of 10 for 6 hours. Cells were fixed, permeabilized and labeled with rabbit-anti-ICP8 367 and rat-anti-Hsc70 antibodies. The inset in the bottom row shows an enlarged image of a VICE domain containing GFP170*.
    Figure Legend Snippet: A model misfolded protein is localized to VICE domains. Vero cells were transfected with 1 ug of plasmid DNA encoding the model misfolded protein GFP170* [2] . Following about 16 hours of protein expression, the cells were infected with KOS at an MOI of 10 for 6 hours. Cells were fixed, permeabilized and labeled with rabbit-anti-ICP8 367 and rat-anti-Hsc70 antibodies. The inset in the bottom row shows an enlarged image of a VICE domain containing GFP170*.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Infection, Labeling

    VICE domains are resistant to detergent extraction. (A) Vero cells were transfected with 1 ug GFP170* [2] . Following 16 hours of protein expression, cells were treated with PBS (left panel) or 0.5% TX-100 detergent extraction buffer (right panel) for 2 minutes on ice. Cells were then fixed in 4% paraformaldehyde, permeabilized in 1% TX-100 and analyzed for localization of GFP170*. (B) Vero cells were mock infected or infected with KOS at an MOI of 10. Following 6 hours of infection, cells were treated with PBS or detergent extraction buffer as described in (A). Hsc70 localization was detected with a rat-anti-Hsc70 (Stressgen) antibody. Imaging was performed on a Zeiss LSM 410 confocal microscope. The gain was set to visualize the diffuse Hsc70, resulting in saturation of the VICE domains.
    Figure Legend Snippet: VICE domains are resistant to detergent extraction. (A) Vero cells were transfected with 1 ug GFP170* [2] . Following 16 hours of protein expression, cells were treated with PBS (left panel) or 0.5% TX-100 detergent extraction buffer (right panel) for 2 minutes on ice. Cells were then fixed in 4% paraformaldehyde, permeabilized in 1% TX-100 and analyzed for localization of GFP170*. (B) Vero cells were mock infected or infected with KOS at an MOI of 10. Following 6 hours of infection, cells were treated with PBS or detergent extraction buffer as described in (A). Hsc70 localization was detected with a rat-anti-Hsc70 (Stressgen) antibody. Imaging was performed on a Zeiss LSM 410 confocal microscope. The gain was set to visualize the diffuse Hsc70, resulting in saturation of the VICE domains.

    Techniques Used: Transfection, Expressing, Infection, Imaging, Microscopy

    25) Product Images from "Strain-Dependent Structural Variants of Herpes Simplex Virus Type 1 ICP34.5 Determine Viral Plaque Size, Efficiency of Glycoprotein Processing, and Viral Release and Neuroinvasive Disease Potential"

    Article Title: Strain-Dependent Structural Variants of Herpes Simplex Virus Type 1 ICP34.5 Determine Viral Plaque Size, Efficiency of Glycoprotein Processing, and Viral Release and Neuroinvasive Disease Potential

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.6.3409-3417.2003

    Ectopic expression of ICP34.5-myc variants determines the tissue culture behaviors of an infecting virus. Vero cells were transfected, and 2 days later they were treated with 1 mg of G418/ml for 3 days to inhibit protein synthesis in untransfected cells and then infected with ICP34.5 deletion mutants ( d 34.5 or TermA) or wild-type viruses (McKrae or AR, a rescue virus from TermA) and harvested 2 days later. Detergent extracts were analyzed by Western blotting. The solid arrows indicate the precursor gC, with an apparent mass of 84 kDa, while the open arrows represent the mature gC, which is 116 kDa. The ratio of the amount of the processed gC to that of precursor gC is indicated below each lane.
    Figure Legend Snippet: Ectopic expression of ICP34.5-myc variants determines the tissue culture behaviors of an infecting virus. Vero cells were transfected, and 2 days later they were treated with 1 mg of G418/ml for 3 days to inhibit protein synthesis in untransfected cells and then infected with ICP34.5 deletion mutants ( d 34.5 or TermA) or wild-type viruses (McKrae or AR, a rescue virus from TermA) and harvested 2 days later. Detergent extracts were analyzed by Western blotting. The solid arrows indicate the precursor gC, with an apparent mass of 84 kDa, while the open arrows represent the mature gC, which is 116 kDa. The ratio of the amount of the processed gC to that of precursor gC is indicated below each lane.

    Techniques Used: Expressing, Transfection, Infection, Western Blot

    26) Product Images from "The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain"

    Article Title: The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain

    Journal: Viruses

    doi: 10.3390/v10100543

    The in vitro characterization of the recombinant iPEDVPT-P96 virus. ( A ) The cytopathic effect in Vero cells infected with the iPEDVPT-P96 virus ( Aa ), PEDVPT-P96 virus ( Ab ), or mock-treated ( Ac ), and the immunocytochemistry ( Ad ) detecting PEDV N protein. The microphotographs were taken at 400× magnification. ( B ) Sequence analysis indicated the appropriate marker mutations of cysteine to thymidine at nucleotide site 24341 and, thymidine to cysteine at nucleotide site 24841, as indicated by the red boxes. ( C ) Plaque morphologies of Vero cells infected with the iPEDVPT-P96 virus, PEDVPT-P96 virus, or mock-infected, acquired after three days of incubation. Note that the iPEDVPT-P96 infection resulted in more consistent plaque sizes. ( D ) The growth kinetics of the iPEDVPT-P96 and PEDVPT-P96 viruses in the Vero cells after infection at 0.01 MOI. The titers of each virus at the indicated time points (hours post-infection; HPI) were expressed as the mean ± standard deviation. Different alphabetic letters indicated significant differences between the iPEDVPT-P96 or PEDVPT-P96 groups ( p
    Figure Legend Snippet: The in vitro characterization of the recombinant iPEDVPT-P96 virus. ( A ) The cytopathic effect in Vero cells infected with the iPEDVPT-P96 virus ( Aa ), PEDVPT-P96 virus ( Ab ), or mock-treated ( Ac ), and the immunocytochemistry ( Ad ) detecting PEDV N protein. The microphotographs were taken at 400× magnification. ( B ) Sequence analysis indicated the appropriate marker mutations of cysteine to thymidine at nucleotide site 24341 and, thymidine to cysteine at nucleotide site 24841, as indicated by the red boxes. ( C ) Plaque morphologies of Vero cells infected with the iPEDVPT-P96 virus, PEDVPT-P96 virus, or mock-infected, acquired after three days of incubation. Note that the iPEDVPT-P96 infection resulted in more consistent plaque sizes. ( D ) The growth kinetics of the iPEDVPT-P96 and PEDVPT-P96 viruses in the Vero cells after infection at 0.01 MOI. The titers of each virus at the indicated time points (hours post-infection; HPI) were expressed as the mean ± standard deviation. Different alphabetic letters indicated significant differences between the iPEDVPT-P96 or PEDVPT-P96 groups ( p

    Techniques Used: In Vitro, Recombinant, Infection, Immunocytochemistry, Sequencing, Marker, Incubation, Standard Deviation

    27) Product Images from "Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene"

    Article Title: Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131129

    Effect of proteasome inhibitor MG132 on the entry of HSV-1 that lacks gC. CHO-nectin-1 cells ( A, B ) or Vero cells ( C ) were treated with MG132 for 15 min at 37°C. HSV-1 KOS, gC∆2–3, 17+, dl 1403, KOS-tk12, or 7134 was added (MOI of ~1) for 6 h. The percent beta-galactosidase activity relative to that obtained in the absence of MG132 is indicated. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.
    Figure Legend Snippet: Effect of proteasome inhibitor MG132 on the entry of HSV-1 that lacks gC. CHO-nectin-1 cells ( A, B ) or Vero cells ( C ) were treated with MG132 for 15 min at 37°C. HSV-1 KOS, gC∆2–3, 17+, dl 1403, KOS-tk12, or 7134 was added (MOI of ~1) for 6 h. The percent beta-galactosidase activity relative to that obtained in the absence of MG132 is indicated. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.

    Techniques Used: Activity Assay

    Resistance of HSV-1 dl 1403 to inhibition by heparin. HSV-1 17+, dl 1403, KOS, or gC∆2–3 (100 PFU per well) was added to Vero cells at 4˚C for 1 hr in the presence of 0 to 5 μg/ml heparin as indicated. Cultures were washed thrice with PBS, and then incubated at 37˚C for 24 hr. Plaque formation was detected by immunoperoxidase staining. Plaque formation in the untreated sample was set to 100%. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.
    Figure Legend Snippet: Resistance of HSV-1 dl 1403 to inhibition by heparin. HSV-1 17+, dl 1403, KOS, or gC∆2–3 (100 PFU per well) was added to Vero cells at 4˚C for 1 hr in the presence of 0 to 5 μg/ml heparin as indicated. Cultures were washed thrice with PBS, and then incubated at 37˚C for 24 hr. Plaque formation was detected by immunoperoxidase staining. Plaque formation in the untreated sample was set to 100%. The data are means of quadruplicate determinations with the standard error of the mean. Results are representative of three independent experiments.

    Techniques Used: Inhibition, Incubation, Immunoperoxidase Staining

    28) Product Images from "Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein"

    Article Title: Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01826-12

    Identification of conformational anti-F monoclonal antibodies (MAbs). (A) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown. FP, fusion peptide; HRA and HRB, heptad repeat regions A and B, respectively; TM, transmembrane domain. The red box represents the position at the C-terminal part of F 2 used for the FLAG epitope insertion. (B and E) Vero (for CDV) or CHO (for MeV) cells were cotransfected with F and H expression plasmids. For IF analysis, cells were stained with the different anti-F MAbs (FLAG, 4941, and 4068 for CDV and FLAG, 186CA, and 19GD6 for MeV) at 24 h posttransfection at 4°C. In some experiments, brief heat shocks (10 min at the indicated temperatures) were performed prior to IF. Alexa Fluor 488-conjugated secondary antibody was then added, and images were captured with a fluorescence confocal microscope (Fluoroview FV1000; Olympus). (C and F) Reactivity of the pair of conformational MAbs with F trimers. Cells were transfected and stained as described in above for panels B and E. Stained cells were then subjected to flow cytometry to record mean fluorescence intensities (MFI). In some experiments, brief heat shocks (10 min at the indicated temperatures) were performed prior to flow cytometry. Means of data from three independent experiments performed in duplicates are shown. (D and G) Virus neutralization assay. A total of 100 TCID 50 units of virus was incubated with the indicated dilution of antibody for 1 h at 37°C. The virus-antibody mixture were then added to Vero cells, and the cells were incubated for 72 h at 37°C. The cells were assayed for luciferase activity for MeV or the number of infectious units for CDV.
    Figure Legend Snippet: Identification of conformational anti-F monoclonal antibodies (MAbs). (A) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown. FP, fusion peptide; HRA and HRB, heptad repeat regions A and B, respectively; TM, transmembrane domain. The red box represents the position at the C-terminal part of F 2 used for the FLAG epitope insertion. (B and E) Vero (for CDV) or CHO (for MeV) cells were cotransfected with F and H expression plasmids. For IF analysis, cells were stained with the different anti-F MAbs (FLAG, 4941, and 4068 for CDV and FLAG, 186CA, and 19GD6 for MeV) at 24 h posttransfection at 4°C. In some experiments, brief heat shocks (10 min at the indicated temperatures) were performed prior to IF. Alexa Fluor 488-conjugated secondary antibody was then added, and images were captured with a fluorescence confocal microscope (Fluoroview FV1000; Olympus). (C and F) Reactivity of the pair of conformational MAbs with F trimers. Cells were transfected and stained as described in above for panels B and E. Stained cells were then subjected to flow cytometry to record mean fluorescence intensities (MFI). In some experiments, brief heat shocks (10 min at the indicated temperatures) were performed prior to flow cytometry. Means of data from three independent experiments performed in duplicates are shown. (D and G) Virus neutralization assay. A total of 100 TCID 50 units of virus was incubated with the indicated dilution of antibody for 1 h at 37°C. The virus-antibody mixture were then added to Vero cells, and the cells were incubated for 72 h at 37°C. The cells were assayed for luciferase activity for MeV or the number of infectious units for CDV.

    Techniques Used: FLAG-tag, Expressing, IF-cells, Staining, Fluorescence, Microscopy, Transfection, Flow Cytometry, Cytometry, Neutralization, Incubation, Luciferase, Activity Assay

    ). Residues flanking the FLAG epitope insertion are highlighted in red. (B) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown. FP, fusion peptide; HRA and HRB, heptad repeat regions A and B, respectively; TM, transmembrane domain. The red boxes represent the positions along the gene selected for FLAG epitope insertions. (C and F) Expression and processing abilities of the different F variants. Total cell protein extraction from the various F mutants expressed in Vero cells was performed. Immunoblots were decorated with a polyclonal anti-HA (MeV F constructs) or polyclonal anti-F (CDV F constructs) antibody. (D) Quantitative fusion assay. Vero-SLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the different F proteins, a plasmid encoding H, and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. Means of data from three independent experiments in duplicate are shown. wt, wild type. (E and G) Syncytium formation assay. Shown are data for cell-cell fusion induction after the cotransfection of Vero-SLAM cells with plasmid DNA encoding various CDV F proteins and H. Representative fields of view were captured at 24 h posttransfection with a fluorescence confocal microscope (Fluoroview FV1000; Olympus).
    Figure Legend Snippet: ). Residues flanking the FLAG epitope insertion are highlighted in red. (B) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown. FP, fusion peptide; HRA and HRB, heptad repeat regions A and B, respectively; TM, transmembrane domain. The red boxes represent the positions along the gene selected for FLAG epitope insertions. (C and F) Expression and processing abilities of the different F variants. Total cell protein extraction from the various F mutants expressed in Vero cells was performed. Immunoblots were decorated with a polyclonal anti-HA (MeV F constructs) or polyclonal anti-F (CDV F constructs) antibody. (D) Quantitative fusion assay. Vero-SLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the different F proteins, a plasmid encoding H, and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. Means of data from three independent experiments in duplicate are shown. wt, wild type. (E and G) Syncytium formation assay. Shown are data for cell-cell fusion induction after the cotransfection of Vero-SLAM cells with plasmid DNA encoding various CDV F proteins and H. Representative fields of view were captured at 24 h posttransfection with a fluorescence confocal microscope (Fluoroview FV1000; Olympus).

    Techniques Used: FLAG-tag, Expressing, Protein Extraction, Western Blot, Construct, Single Vesicle Fusion Assay, Infection, Transfection, Plasmid Preparation, Luciferase, Tube Formation Assay, Cotransfection, Fluorescence, Microscopy

    29) Product Images from "Analysis of the Role of Autophagy in Replication of Herpes Simplex Virus in Cell Culture ▿"

    Article Title: Analysis of the Role of Autophagy in Replication of Herpes Simplex Virus in Cell Culture ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01356-07

    Autophagy does not significantly affect HSV-1 growth. Primary Atg5 −/− and Atg5 +/+ MEFs were infected at a multiplicity of 0.01 PFU/cell. Cells and supernatants were collected at the indicated times postinfection, and titers were determined on Vero cells. Results shown represent data collected from three independent experiments. Data points represent the geometric mean number of PFU/ml of material in which titers were determined ± the standard error of the mean for three samples per virus per time point. The growth of 17termAR was significantly different ( P
    Figure Legend Snippet: Autophagy does not significantly affect HSV-1 growth. Primary Atg5 −/− and Atg5 +/+ MEFs were infected at a multiplicity of 0.01 PFU/cell. Cells and supernatants were collected at the indicated times postinfection, and titers were determined on Vero cells. Results shown represent data collected from three independent experiments. Data points represent the geometric mean number of PFU/ml of material in which titers were determined ± the standard error of the mean for three samples per virus per time point. The growth of 17termAR was significantly different ( P

    Techniques Used: Infection

    30) Product Images from "B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases"

    Article Title: B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02542

    B/αGC/vacESAT6 up-regulates co-stimulatory molecules on B cells. (A,B) Vero cells were transduced with vaccinia-ESAT6 (vacESAT6) at a multiplicity of infection (MOI) of 1. Transduced cells were fluorescently stained for ESAT6 (green) and counterstained with DAPI (blue) for nuclei which were analyzed by confocal microscopy to detect the expression of ESAT6 (scale bar = 20 μm). (A) Representative confocal images and (B) Representation of the evaluation of green fluorescent area. (C,D) B220 + cells were isolated from splenocytes of naïve C57BL/6 mice. Isolated B220 + cells were transduced with vacESAT6 at a MOI of 1 and/or loaded with 1 μg/ml of αGC and then co-cultured with naïve splenocytes for 24 h. Incubated cells were stained to examine the expression of B220, CD40, and CD86. Expression levels of CD40 and CD86 in B220 + cells were analyzed by flow cytometry. (C) Representative flow cytometry histogram and (D) summary of mean fluorescence intensity of CD40 and CD86 expression in B cells. ANOVA. * p
    Figure Legend Snippet: B/αGC/vacESAT6 up-regulates co-stimulatory molecules on B cells. (A,B) Vero cells were transduced with vaccinia-ESAT6 (vacESAT6) at a multiplicity of infection (MOI) of 1. Transduced cells were fluorescently stained for ESAT6 (green) and counterstained with DAPI (blue) for nuclei which were analyzed by confocal microscopy to detect the expression of ESAT6 (scale bar = 20 μm). (A) Representative confocal images and (B) Representation of the evaluation of green fluorescent area. (C,D) B220 + cells were isolated from splenocytes of naïve C57BL/6 mice. Isolated B220 + cells were transduced with vacESAT6 at a MOI of 1 and/or loaded with 1 μg/ml of αGC and then co-cultured with naïve splenocytes for 24 h. Incubated cells were stained to examine the expression of B220, CD40, and CD86. Expression levels of CD40 and CD86 in B220 + cells were analyzed by flow cytometry. (C) Representative flow cytometry histogram and (D) summary of mean fluorescence intensity of CD40 and CD86 expression in B cells. ANOVA. * p

    Techniques Used: Transduction, Infection, Staining, Confocal Microscopy, Expressing, Isolation, Mouse Assay, Cell Culture, Incubation, Flow Cytometry, Cytometry, Fluorescence

    31) Product Images from "Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation"

    Article Title: Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02733-12

    Mutagenesis analysis of the VPg-3D pol interface on EV71 replication. Results for the mutants of the 3D pol protein (A) and VPg (B) are shown. (Top) Vero cells were transfected with WT and mutant genome-length RNAs (5 μg) and analyzed for viral VP1 protein expression by IFA 48 h p.t.; (bottom) the plaque morphologies of WT and mutant viruses are also shown. N.D., not detectable.
    Figure Legend Snippet: Mutagenesis analysis of the VPg-3D pol interface on EV71 replication. Results for the mutants of the 3D pol protein (A) and VPg (B) are shown. (Top) Vero cells were transfected with WT and mutant genome-length RNAs (5 μg) and analyzed for viral VP1 protein expression by IFA 48 h p.t.; (bottom) the plaque morphologies of WT and mutant viruses are also shown. N.D., not detectable.

    Techniques Used: Mutagenesis, Transfection, Expressing, Immunofluorescence

    32) Product Images from "Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation"

    Article Title: Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.018

    A regulated interaction between UL16 and the gD cytoplasmic tail. ( A ) Vero cells were singly transfected (top row) with plasmids that express full-length UL16-GFP, UL16 NTD-GFP, or UL16 CTD-GFP. Additionally, each of these constructs were co-expressed with gD-HA (bottom three rows). Cells were fixed, permeabilized, and stained with a monoclonal antibody against the HA tag at one day post-transfection. ( B ) Purified His 6 -UL16(1–155) protein was incubated with the indicated GST-fusion proteins either in the presence or absence of NEM bound to glutathione-sepharose beads in 0.5% NP-40 buffer at 37°C. The beads were then washed, boiled in sample buffer, and the proteins were analyzed by western blotting.
    Figure Legend Snippet: A regulated interaction between UL16 and the gD cytoplasmic tail. ( A ) Vero cells were singly transfected (top row) with plasmids that express full-length UL16-GFP, UL16 NTD-GFP, or UL16 CTD-GFP. Additionally, each of these constructs were co-expressed with gD-HA (bottom three rows). Cells were fixed, permeabilized, and stained with a monoclonal antibody against the HA tag at one day post-transfection. ( B ) Purified His 6 -UL16(1–155) protein was incubated with the indicated GST-fusion proteins either in the presence or absence of NEM bound to glutathione-sepharose beads in 0.5% NP-40 buffer at 37°C. The beads were then washed, boiled in sample buffer, and the proteins were analyzed by western blotting.

    Techniques Used: Transfection, Construct, Staining, Purification, Incubation, Western Blot

    The UL16-gD interaction is not critical for the gBsyn phenotype. ( A ) Cells were infected with gBsyn, M6/syn, ΔCT/syn, or ΔCT.R/syn at a low MOI. Images were taken with an inverted light microscope at 2–3 days post infection. ( B ) Vero cells were infected with the indicated viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion and the cells were collected. Samples were analyzed by western blotting. (C) Duplicate cultures of Vero cells were infected with the indicated viruses at an MOI of 0.1. The media containing extracellular virions was collected, and the infected cells were harvested and processed separately at 6-hour time points. Infectious virus was titered by plaque assay. ( D ) Purified His 6 -UL16 was incubated in 0.5% NP-40 buffer at 37°C for 5 hr with glutathione-sepharose beads bearing GST fusion proteins with the entire tail of gD or truncated sections of the tail. GST-UL11 and GST alone served as respective positive and negative controls. After incubation, the beads were washed, boiled in sample buffer, and the presence of bound His 6 -UL16 was analyzed by western blotting.
    Figure Legend Snippet: The UL16-gD interaction is not critical for the gBsyn phenotype. ( A ) Cells were infected with gBsyn, M6/syn, ΔCT/syn, or ΔCT.R/syn at a low MOI. Images were taken with an inverted light microscope at 2–3 days post infection. ( B ) Vero cells were infected with the indicated viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion and the cells were collected. Samples were analyzed by western blotting. (C) Duplicate cultures of Vero cells were infected with the indicated viruses at an MOI of 0.1. The media containing extracellular virions was collected, and the infected cells were harvested and processed separately at 6-hour time points. Infectious virus was titered by plaque assay. ( D ) Purified His 6 -UL16 was incubated in 0.5% NP-40 buffer at 37°C for 5 hr with glutathione-sepharose beads bearing GST fusion proteins with the entire tail of gD or truncated sections of the tail. GST-UL11 and GST alone served as respective positive and negative controls. After incubation, the beads were washed, boiled in sample buffer, and the presence of bound His 6 -UL16 was analyzed by western blotting.

    Techniques Used: Infection, Light Microscopy, Western Blot, Plaque Assay, Purification, Incubation

    UL16 interacts with the cytoplasmic tail of gD. ( A ) Vero cells were infected with WT, ΔUL11, or ΔUL16 viruses. At 24 hr post infection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After 5 hr of incubation at room temperature or at 37°C, the beads were washed, boiled in sample buffer, and the proteins were analyzed by western blotting with an antibody specific for UL16. ( B ) Vero cells were transfected with expression plasmids for UL16-GFP or GFP-only. At 18–24 hr post transfection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gE.CT, GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After incubation, the beads were processed as in (A) and an antibody specific for GFP was used. ( C ) Increasing amounts of purified His 6 -UL16 produced in E. coli was incubated with GST-gD.CT, GST-gE.CT, or GST-only bound to glutathionesepharose beads in 0.5% NP-40 buffer at 37°C. After incubation, the beads were processed as in (A), a Ponceau stain for total protein was performed, and an antibody specific for the His tag was used to probe the western blot.
    Figure Legend Snippet: UL16 interacts with the cytoplasmic tail of gD. ( A ) Vero cells were infected with WT, ΔUL11, or ΔUL16 viruses. At 24 hr post infection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After 5 hr of incubation at room temperature or at 37°C, the beads were washed, boiled in sample buffer, and the proteins were analyzed by western blotting with an antibody specific for UL16. ( B ) Vero cells were transfected with expression plasmids for UL16-GFP or GFP-only. At 18–24 hr post transfection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gE.CT, GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After incubation, the beads were processed as in (A) and an antibody specific for GFP was used. ( C ) Increasing amounts of purified His 6 -UL16 produced in E. coli was incubated with GST-gD.CT, GST-gE.CT, or GST-only bound to glutathionesepharose beads in 0.5% NP-40 buffer at 37°C. After incubation, the beads were processed as in (A), a Ponceau stain for total protein was performed, and an antibody specific for the His tag was used to probe the western blot.

    Techniques Used: Infection, Incubation, Purification, Staining, Western Blot, Transfection, Expressing, Produced

    UL16 is critical for gD packaging. Vero cells were infected with WT, UL16-null mutant, or repaired (ΔUL16.R or ΔUL16.Rev35) viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion, and the collected cells were lysed with SDS-PAGE loading buffer. Samples were analyzed by western blotting with antibodies specific for the indicated viral proteins.
    Figure Legend Snippet: UL16 is critical for gD packaging. Vero cells were infected with WT, UL16-null mutant, or repaired (ΔUL16.R or ΔUL16.Rev35) viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion, and the collected cells were lysed with SDS-PAGE loading buffer. Samples were analyzed by western blotting with antibodies specific for the indicated viral proteins.

    Techniques Used: Infection, Mutagenesis, SDS Page, Western Blot

    33) Product Images from "The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication"

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    Journal: Virology Journal

    doi: 10.1186/1743-422X-10-205

    Effect of eIF3L on yellow fever virus replication. ( A ) eIF3L overexpression in BHK21-rep-YFV17D LucNeoIres cells at 48 h post-transfection using an anti-Flag antibody or anti-GAPDH antibody as a control. eIF-pCDNAFlag, overexpressing eIF3L; pCDNAFlag, empty vector control; MOCK, control cells. ( B ) Luciferase activity at 48 h transfection with eIF3L in BHK21-rep-YFV17D LucNeoIres cell lysates; MOCK, control cells; pCDNAFlag, empty vector control; eIF3L-pCDNAFlag, eIF3L overexpression; siRNA NS1, positive control. *Significantly different between the RNAi NS1 and mock samples; **significantly different between the pCDNAFlag-eIF3L and mock samples. ( C ) Quantification of viral RNA transcripts at 24 and 48 h post-infection with pseudotyped lentiviral particles using real-time PCR analysis. pLKO.1-eIF3LshRNA1, eIF3L silenced and infected cells; pLKO.1-eIF3LshRNA2, eIF3L silenced and infected cells; pLKO, empty vector control-infected cells; non-target, plasmid control non-target infected cells; YFV, infected cells; MOCK, control cells. ( D ) The plaque reduction assay indicates a discrete reduction in viral replication. ( E ) Quantification of viral RNA in Vero cell supernatants overexpressing eIF3L at 6, 12, 24, and 48 h post-infection.
    Figure Legend Snippet: Effect of eIF3L on yellow fever virus replication. ( A ) eIF3L overexpression in BHK21-rep-YFV17D LucNeoIres cells at 48 h post-transfection using an anti-Flag antibody or anti-GAPDH antibody as a control. eIF-pCDNAFlag, overexpressing eIF3L; pCDNAFlag, empty vector control; MOCK, control cells. ( B ) Luciferase activity at 48 h transfection with eIF3L in BHK21-rep-YFV17D LucNeoIres cell lysates; MOCK, control cells; pCDNAFlag, empty vector control; eIF3L-pCDNAFlag, eIF3L overexpression; siRNA NS1, positive control. *Significantly different between the RNAi NS1 and mock samples; **significantly different between the pCDNAFlag-eIF3L and mock samples. ( C ) Quantification of viral RNA transcripts at 24 and 48 h post-infection with pseudotyped lentiviral particles using real-time PCR analysis. pLKO.1-eIF3LshRNA1, eIF3L silenced and infected cells; pLKO.1-eIF3LshRNA2, eIF3L silenced and infected cells; pLKO, empty vector control-infected cells; non-target, plasmid control non-target infected cells; YFV, infected cells; MOCK, control cells. ( D ) The plaque reduction assay indicates a discrete reduction in viral replication. ( E ) Quantification of viral RNA in Vero cell supernatants overexpressing eIF3L at 6, 12, 24, and 48 h post-infection.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Positive Control, Infection, Real-time Polymerase Chain Reaction

    Coimmunoprecipitation of YFV NS5 and eIF3L in mammalian cells. Vero cells were transfected with a plasmid expressing Flag-tagged eIF3L and infected with YFV strain 17D at an M.O.I. of 5. At 48 h post-infection, the cells were lysed and subjected to immunoprecipitation using anti-Flag M2 affinity gel beads. The coimmunoprecipitated proteins were analyzed by a western blot assay using an anti-YFNS5 antibody. ( A ) Cell lysates immunoprecipitated using the anti-Flag antibody. Input, positive control eIF3L (67 kDa) from transfected cells; eIF3L+YFV, eIF3L immunoprecipitated from transfected and infected cells; YFV, infected and non-transfected cells; MOCK, non-infected and non-transfected cells. ( B ) Coimmunoprecipitation of the NS5 protein with the anti-YFV NS5 antibody. Input, positive control YFV NS5 from infected cells (98 kDa); eIF3L+YFV, coimmunoprecipitation of YFV NS5 with Flag-eIF3L showing a positive interaction; YFV, MOCK, YFV NS5 was not precipitated in non-transfected cells.
    Figure Legend Snippet: Coimmunoprecipitation of YFV NS5 and eIF3L in mammalian cells. Vero cells were transfected with a plasmid expressing Flag-tagged eIF3L and infected with YFV strain 17D at an M.O.I. of 5. At 48 h post-infection, the cells were lysed and subjected to immunoprecipitation using anti-Flag M2 affinity gel beads. The coimmunoprecipitated proteins were analyzed by a western blot assay using an anti-YFNS5 antibody. ( A ) Cell lysates immunoprecipitated using the anti-Flag antibody. Input, positive control eIF3L (67 kDa) from transfected cells; eIF3L+YFV, eIF3L immunoprecipitated from transfected and infected cells; YFV, infected and non-transfected cells; MOCK, non-infected and non-transfected cells. ( B ) Coimmunoprecipitation of the NS5 protein with the anti-YFV NS5 antibody. Input, positive control YFV NS5 from infected cells (98 kDa); eIF3L+YFV, coimmunoprecipitation of YFV NS5 with Flag-eIF3L showing a positive interaction; YFV, MOCK, YFV NS5 was not precipitated in non-transfected cells.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Infection, Immunoprecipitation, Western Blot, Positive Control

    34) Product Images from "Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells"

    Article Title: Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

    Journal: Virology Journal

    doi: 10.1186/1743-422X-11-150

    Comparison of the replication kinetics of MODV-WNV(prM-E), MODV and WNV in Vero cells. Subconfluent monolayers of Vero cells were inoculated with MODV-WNV(prM-E), MODV and WNV at a m.o.i of 0.1. Supernatants were collected daily for 7 days and tested by plaque assay. MODV-WNV(prM-E) had been passaged once in BHK-21 cells and once in Vero cells prior to the experiments.
    Figure Legend Snippet: Comparison of the replication kinetics of MODV-WNV(prM-E), MODV and WNV in Vero cells. Subconfluent monolayers of Vero cells were inoculated with MODV-WNV(prM-E), MODV and WNV at a m.o.i of 0.1. Supernatants were collected daily for 7 days and tested by plaque assay. MODV-WNV(prM-E) had been passaged once in BHK-21 cells and once in Vero cells prior to the experiments.

    Techniques Used: Plaque Assay

    Comparison of the plaque morphologies of MODV-WNV(prM-E) and the parental viruses in Vero cells. Confluent monolayers of Vero cells in six-well plates were inoculated with MODV-WNV(prM-E), MODV or WNV. Cells were fixed and plaques were visualized by staining with crystal violet at 3, 5 and 7 days p.i. Images were transferred into Microsoft Photoshop and plaque diameters were measured. The chimeric virus had been passaged one in BHK-21 cells and twice in Vero cells prior to this experiment.
    Figure Legend Snippet: Comparison of the plaque morphologies of MODV-WNV(prM-E) and the parental viruses in Vero cells. Confluent monolayers of Vero cells in six-well plates were inoculated with MODV-WNV(prM-E), MODV or WNV. Cells were fixed and plaques were visualized by staining with crystal violet at 3, 5 and 7 days p.i. Images were transferred into Microsoft Photoshop and plaque diameters were measured. The chimeric virus had been passaged one in BHK-21 cells and twice in Vero cells prior to this experiment.

    Techniques Used: Staining

    35) Product Images from "Inhibition of the Host Translation Shutoff Response by Herpes Simplex Virus 1 Triggers Nuclear Envelope-Derived Autophagy"

    Article Title: Inhibition of the Host Translation Shutoff Response by Herpes Simplex Virus 1 Triggers Nuclear Envelope-Derived Autophagy

    Journal: Journal of Virology

    doi: 10.1128/JVI.02974-12

    NEDA is rescued by gH overexpression. Vero cells, Vero CR1 cells that express the late HSV-1 protein gH (Vero-gH), or Vero E5 cells that express the immediate early protein ICP4 (Vero-ICP4) were infected with the HSV-1 wt or HSV-1 ΔPP1α
    Figure Legend Snippet: NEDA is rescued by gH overexpression. Vero cells, Vero CR1 cells that express the late HSV-1 protein gH (Vero-gH), or Vero E5 cells that express the immediate early protein ICP4 (Vero-ICP4) were infected with the HSV-1 wt or HSV-1 ΔPP1α

    Techniques Used: Over Expression, Infection

    36) Product Images from "Reverse Genetics System for Rabbit vesivirus"

    Article Title: Reverse Genetics System for Rabbit vesivirus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.596245

    Microscope images (100 X) of 293T cells either (A) infected with rFPV-T7 and further transfected with DNA infectious clones or (B) transfected with capped or uncapped synthetic genome-length viral RNA. GFP expression from pT7-GFP plasmid visualized using a fluorescence filter served as a transfection efficiency control. (C) Bright field microscope images (40 X) of Vero cells infected with either wtRaV, rRaV, or rRaV/Xh (MOI = 0.1, passage 3). The photos were taken at 6, 12, and 24 hpi. (D) Morphology of wild-type and rescued RaV lysis plaques in Vero cells under 1% agarose overlay.
    Figure Legend Snippet: Microscope images (100 X) of 293T cells either (A) infected with rFPV-T7 and further transfected with DNA infectious clones or (B) transfected with capped or uncapped synthetic genome-length viral RNA. GFP expression from pT7-GFP plasmid visualized using a fluorescence filter served as a transfection efficiency control. (C) Bright field microscope images (40 X) of Vero cells infected with either wtRaV, rRaV, or rRaV/Xh (MOI = 0.1, passage 3). The photos were taken at 6, 12, and 24 hpi. (D) Morphology of wild-type and rescued RaV lysis plaques in Vero cells under 1% agarose overlay.

    Techniques Used: Microscopy, Infection, Transfection, Clone Assay, Expressing, Plasmid Preparation, Fluorescence, Lysis

    (A) One-step growth curves of wtRaV and rRaV/Xh in Vero cells infected at MOI = 10 and incubated for 14 h. No significant statistical differences were found between both viral titers within each time point (Student’s t -tests, p > 0.05). (B) Effect of the DNA amount used in transfection (passage 0) over the titer of viruses recovered in passage 1 supernatants. Transfections were performed in 6-well plates with either 1, 3, or 5 μg of the infectious cDNA clone. For each DNA quantity used in passage 0, plaque assay wells inoculated with the corresponding passage 1 supernatant dilutions 10 –3 and 10 –4 are shown.
    Figure Legend Snippet: (A) One-step growth curves of wtRaV and rRaV/Xh in Vero cells infected at MOI = 10 and incubated for 14 h. No significant statistical differences were found between both viral titers within each time point (Student’s t -tests, p > 0.05). (B) Effect of the DNA amount used in transfection (passage 0) over the titer of viruses recovered in passage 1 supernatants. Transfections were performed in 6-well plates with either 1, 3, or 5 μg of the infectious cDNA clone. For each DNA quantity used in passage 0, plaque assay wells inoculated with the corresponding passage 1 supernatant dilutions 10 –3 and 10 –4 are shown.

    Techniques Used: Infection, Incubation, Transfection, Plaque Assay

    37) Product Images from "A Structurally Unresolved Head Segment of Defined Length Favors Proper Measles Virus Hemagglutinin Tetramerization and Efficient Membrane Fusion Triggering"

    Article Title: A Structurally Unresolved Head Segment of Defined Length Favors Proper Measles Virus Hemagglutinin Tetramerization and Efficient Membrane Fusion Triggering

    Journal: Journal of Virology

    doi: 10.1128/JVI.02253-15

    Protein expression levels of unresolved head segment mutants. (A) Total protein expression. Cytoplasmic extracts of Vero cells transfected with the H expression plasmids indicated below the gel were separated by 4 to 15% SDS-PAGE under reducing conditions and immunoblotted with an anti-H (cytoplasmic tail) antibody. M, H-monomer band. A, β-actin was used as a loading control. (B) Cell surface protein expression. 293T cells transfected with the indicated H construct were stained with a blend of two anti-H ectodomain antibodies and phycoerythrin-conjugated secondary antibody. The mean fluorescence intensity of each mutant is presented as a percentage of the standard H-protein levels. Error bars indicate standard deviations from three experiments.
    Figure Legend Snippet: Protein expression levels of unresolved head segment mutants. (A) Total protein expression. Cytoplasmic extracts of Vero cells transfected with the H expression plasmids indicated below the gel were separated by 4 to 15% SDS-PAGE under reducing conditions and immunoblotted with an anti-H (cytoplasmic tail) antibody. M, H-monomer band. A, β-actin was used as a loading control. (B) Cell surface protein expression. 293T cells transfected with the indicated H construct were stained with a blend of two anti-H ectodomain antibodies and phycoerythrin-conjugated secondary antibody. The mean fluorescence intensity of each mutant is presented as a percentage of the standard H-protein levels. Error bars indicate standard deviations from three experiments.

    Techniques Used: Expressing, Transfection, SDS Page, Construct, Staining, Fluorescence, Mutagenesis

    Higher-order H oligomers of two deletion mutants are efficiently transported to the cell surface. Vero cells transfected with the indicated expression plasmids were lysed at 24 hours posttransfection. Biotin-labeled surface proteins (left) or total cell protein extracts (right) were separated on nonreducing SDS-PAGE and subjected to anti-H-protein immunoblotting (top gel). The same extracts were then blotted for β-actin (bottom gel). Molecular mass markers are indicated on the left side. O, stable higher-order H oligomers; D, dimers; M, monomers; A, β-actin.
    Figure Legend Snippet: Higher-order H oligomers of two deletion mutants are efficiently transported to the cell surface. Vero cells transfected with the indicated expression plasmids were lysed at 24 hours posttransfection. Biotin-labeled surface proteins (left) or total cell protein extracts (right) were separated on nonreducing SDS-PAGE and subjected to anti-H-protein immunoblotting (top gel). The same extracts were then blotted for β-actin (bottom gel). Molecular mass markers are indicated on the left side. O, stable higher-order H oligomers; D, dimers; M, monomers; A, β-actin.

    Techniques Used: Transfection, Expressing, Labeling, SDS Page

    Oligomeric states of the mutants. (A) Cytoplasmic extracts of Vero cells transfected with the indicated H expression plasmid were separated by 4 to 15% SDS-PAGE under nonreducing conditions and immunoblotted with an anti-H (cytoplasmic tail) antibody. The different H forms are indicated. O, stable higher-order H oligomers; D, dimers; M, monomers. A, β-actin, used as a loading control.
    Figure Legend Snippet: Oligomeric states of the mutants. (A) Cytoplasmic extracts of Vero cells transfected with the indicated H expression plasmid were separated by 4 to 15% SDS-PAGE under nonreducing conditions and immunoblotted with an anti-H (cytoplasmic tail) antibody. The different H forms are indicated. O, stable higher-order H oligomers; D, dimers; M, monomers. A, β-actin, used as a loading control.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, SDS Page

    38) Product Images from "Cell-type apoptosis in lung during SARS-CoV-2 infection"

    Article Title: Cell-type apoptosis in lung during SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.12.23.424254

    Co-culture of Vero cells and relatively nonpermissive HUVECs results in apoptosis following SARS-CoV-2 infection. HUVECs (in the culture wells) were co-cultured with or without Vero cells (in the culture insert). The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 72 hours. Fixed HUVECs in the well were subjected to IF staining to detect SARS-CoV-2 (red) or CD31 (red; an EC-specific marker) and TUNEL (green). Fixed Vero cells in the insert were subjected to IF staining to detect SARS-CoV-2 and TUNEL ( Fig. S2 ). Nuclei of HUVECs were counterstained with DAPI (blue) Scalebars, 20 µm.
    Figure Legend Snippet: Co-culture of Vero cells and relatively nonpermissive HUVECs results in apoptosis following SARS-CoV-2 infection. HUVECs (in the culture wells) were co-cultured with or without Vero cells (in the culture insert). The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 72 hours. Fixed HUVECs in the well were subjected to IF staining to detect SARS-CoV-2 (red) or CD31 (red; an EC-specific marker) and TUNEL (green). Fixed Vero cells in the insert were subjected to IF staining to detect SARS-CoV-2 and TUNEL ( Fig. S2 ). Nuclei of HUVECs were counterstained with DAPI (blue) Scalebars, 20 µm.

    Techniques Used: Co-Culture Assay, Infection, Cell Culture, Staining, Marker, TUNEL Assay

    Co-culture of Vero cells and relatively nonpermissive BEAS2B cells results in apoptosis following SARS-CoV-2 infection. BEAS2B (in the wells) were co-cultured with or without Vero cells (in the insert). The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 72 hours. Fixed BEAS2B cells in the well were subjected to IF staining to detect SARS-CoV-2 (red) and TUNEL (green). Fixed Vero cells in the insert were subjected to IF staining to detect SARS-CoV-2 and TUNEL ( Fig. S2 ). Nuclei of BEAS2B cells were counterstained with DAPI (blue) Scalebars, 20 µm.
    Figure Legend Snippet: Co-culture of Vero cells and relatively nonpermissive BEAS2B cells results in apoptosis following SARS-CoV-2 infection. BEAS2B (in the wells) were co-cultured with or without Vero cells (in the insert). The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 72 hours. Fixed BEAS2B cells in the well were subjected to IF staining to detect SARS-CoV-2 (red) and TUNEL (green). Fixed Vero cells in the insert were subjected to IF staining to detect SARS-CoV-2 and TUNEL ( Fig. S2 ). Nuclei of BEAS2B cells were counterstained with DAPI (blue) Scalebars, 20 µm.

    Techniques Used: Co-Culture Assay, Infection, Cell Culture, Staining, TUNEL Assay

    Pharmacological activators of EPAC protect ECs from apoptosis in Vero cells and HUVECs co-cultures following SARS-CoV-2 infection. HUVECs were co-cultured with Vero cells. The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 24 hours before treatment with I942 (5µM), NY0173 (5µM) and vehicle for 48 hours. ( A ) Fixed HUVECs in the well were subjected to IF staining for SARS-CoV-2 (red) and TUNEL assay (green). Fixed Vero cells in the inserts were subjected to IF staining to for SARS-CoV-2 and TUNEL assay ( Fig. S3 ). Nuclei of HUVECs were counterstained with DAPI (blue). Scale bars, 100 µm. ( B ) Extent of TUNEL signal-positive HUVECs is normalized to total nuclear content (DAPI staining) in each filed. The data presented are representative of three independent experiments (n=78 in each group). * compared to the vehicle group, P
    Figure Legend Snippet: Pharmacological activators of EPAC protect ECs from apoptosis in Vero cells and HUVECs co-cultures following SARS-CoV-2 infection. HUVECs were co-cultured with Vero cells. The co-cultures were exposed to 0.1 MOI of SARS-CoV-2 for 24 hours before treatment with I942 (5µM), NY0173 (5µM) and vehicle for 48 hours. ( A ) Fixed HUVECs in the well were subjected to IF staining for SARS-CoV-2 (red) and TUNEL assay (green). Fixed Vero cells in the inserts were subjected to IF staining to for SARS-CoV-2 and TUNEL assay ( Fig. S3 ). Nuclei of HUVECs were counterstained with DAPI (blue). Scale bars, 100 µm. ( B ) Extent of TUNEL signal-positive HUVECs is normalized to total nuclear content (DAPI staining) in each filed. The data presented are representative of three independent experiments (n=78 in each group). * compared to the vehicle group, P

    Techniques Used: Infection, Cell Culture, Staining, TUNEL Assay

    SARS-CoV-2 infection-induced apoptosis in Vero cells. ( A-C ) Uninfected controls. ( D-F ) TUNEL signals (green) detected in Vero cells at 72 hours p.i. with 0.1 MOI SARS-CoV-2 (red). Nuclei of cells counterstained with DAPI (blue). Scalebars, 20 µm. ( G ) Western blotting shows SARS-CoV-2 infection triggered cleavages of PARP and caspase-9 at 72 hours p.i. in Vero cells, but not in HUVECs.
    Figure Legend Snippet: SARS-CoV-2 infection-induced apoptosis in Vero cells. ( A-C ) Uninfected controls. ( D-F ) TUNEL signals (green) detected in Vero cells at 72 hours p.i. with 0.1 MOI SARS-CoV-2 (red). Nuclei of cells counterstained with DAPI (blue). Scalebars, 20 µm. ( G ) Western blotting shows SARS-CoV-2 infection triggered cleavages of PARP and caspase-9 at 72 hours p.i. in Vero cells, but not in HUVECs.

    Techniques Used: Infection, TUNEL Assay, Western Blot

    39) Product Images from "The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2"

    Article Title: The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2

    Journal: Journal of Virology

    doi: 10.1128/JVI.01246-20

    MERS-CoV ExoN knockout mutants are nonviable. (A) Phenotype of MERS-CoV nsp14 ExoN mutants used in this study, scored at 2 dpt. (B) Comparison of plaque phenotype of selected ExoN mutants in HuH7 cells. Plaque assays were performed using supernatants harvested from transfected cell cultures at 3 dpt, which were diluted 10 −4 for wt and mutant E191D and used in undiluted form for the D90A/E92A ExoN knockout double mutant (DM) and the H229C ZF1 mutant. (C) Immunolabeling (2 dpt) of cell cultures consisting of a mixture of (nonsusceptible) BHK-21 cells transfected with in vitro -made full-length MERS-CoV RNA and susceptible (DPP4-expressing) Vero cells used to amplify any infectious progeny released from the transfected BHK-21 cells. (Left) wt virus; (middle) DM mutant; (right) H229C mutant. Cells were labeled for dsRNA (green) and nsp4 (red). Bar, 100 μm.
    Figure Legend Snippet: MERS-CoV ExoN knockout mutants are nonviable. (A) Phenotype of MERS-CoV nsp14 ExoN mutants used in this study, scored at 2 dpt. (B) Comparison of plaque phenotype of selected ExoN mutants in HuH7 cells. Plaque assays were performed using supernatants harvested from transfected cell cultures at 3 dpt, which were diluted 10 −4 for wt and mutant E191D and used in undiluted form for the D90A/E92A ExoN knockout double mutant (DM) and the H229C ZF1 mutant. (C) Immunolabeling (2 dpt) of cell cultures consisting of a mixture of (nonsusceptible) BHK-21 cells transfected with in vitro -made full-length MERS-CoV RNA and susceptible (DPP4-expressing) Vero cells used to amplify any infectious progeny released from the transfected BHK-21 cells. (Left) wt virus; (middle) DM mutant; (right) H229C mutant. Cells were labeled for dsRNA (green) and nsp4 (red). Bar, 100 μm.

    Techniques Used: Knock-Out, Transfection, Mutagenesis, Immunolabeling, In Vitro, Expressing, Labeling

    A SARS-CoV-2 ExoN knockout mutant is nonviable. (A) Plaque phenotypes of wt SARS-CoV (left) and SARS-CoV-2 (right) and their corresponding ExoN motif I knockout double mutants (DM; D90A/E92A) in Vero E6 cells. Plaque assays were performed using supernatants harvested from transfected cell cultures at 2 dpt for SARS-CoV and 3 dpt for SARS-CoV-2. Samples were diluted 10 −6 for SARS-CoV wt, 10 −5 for SARS-CoV-2 wt and SARS-CoV DM, and 10 −1 for the SARS-CoV-2 DM mutant. (B) Immunolabeling (2 dpt) as described for Fig. 2C but with Vero E6 cells for amplification of SARS-CoV-2 progeny released from BHK-21 cells transfected with wt (top) or DM (bottom) full-length RNA. Bar, 100 μm.
    Figure Legend Snippet: A SARS-CoV-2 ExoN knockout mutant is nonviable. (A) Plaque phenotypes of wt SARS-CoV (left) and SARS-CoV-2 (right) and their corresponding ExoN motif I knockout double mutants (DM; D90A/E92A) in Vero E6 cells. Plaque assays were performed using supernatants harvested from transfected cell cultures at 2 dpt for SARS-CoV and 3 dpt for SARS-CoV-2. Samples were diluted 10 −6 for SARS-CoV wt, 10 −5 for SARS-CoV-2 wt and SARS-CoV DM, and 10 −1 for the SARS-CoV-2 DM mutant. (B) Immunolabeling (2 dpt) as described for Fig. 2C but with Vero E6 cells for amplification of SARS-CoV-2 progeny released from BHK-21 cells transfected with wt (top) or DM (bottom) full-length RNA. Bar, 100 μm.

    Techniques Used: Knock-Out, Mutagenesis, Transfection, Immunolabeling, Amplification

    Impact of ExoN inactivation on intracellular MERS-CoV RNA synthesis. In-gel hybridization analysis of intracellular RNA isolated after 2 or 3 dpt of transfected BHK-21 cells, which were subsequently mixed with HuH7 or Vero cells, as indicated. Purified RNA was separated in an agarose gel and probed with a radiolabeled oligonucleotide probe recognizing the MERS-CoV genome and subgenomic mRNAs.
    Figure Legend Snippet: Impact of ExoN inactivation on intracellular MERS-CoV RNA synthesis. In-gel hybridization analysis of intracellular RNA isolated after 2 or 3 dpt of transfected BHK-21 cells, which were subsequently mixed with HuH7 or Vero cells, as indicated. Purified RNA was separated in an agarose gel and probed with a radiolabeled oligonucleotide probe recognizing the MERS-CoV genome and subgenomic mRNAs.

    Techniques Used: Hybridization, Isolation, Transfection, Purification, Agarose Gel Electrophoresis

    Characterization of growth kinetics of rMERS-nsp14-E191D and its sensitivity to 5-FU treatment. Vero cells (A) or HuH7 cells (B) were infected at an MOI of 3, supernatant was harvested at the indicated time points, and viral progeny titers were measured by plaque assay from two independent experiments using duplicates ( n = 4; values are means ± standard deviations [SD]). (C) Plaque phenotype in HuH7 cells of rMERS-CoV nsp14-E191D and wt control in the absence or presence of the mutagenic agent 5-FU. (D) Dose-response curve of wt and E191D mutant MERS-CoV in the presence of 5-FU concentrations up to 400 μM (MOI, 0.1; n = 4; means ± SD). Statistical significance of the difference with wt virus at each time point (A and B) or concentration (D) was assessed by paired t test. *, P
    Figure Legend Snippet: Characterization of growth kinetics of rMERS-nsp14-E191D and its sensitivity to 5-FU treatment. Vero cells (A) or HuH7 cells (B) were infected at an MOI of 3, supernatant was harvested at the indicated time points, and viral progeny titers were measured by plaque assay from two independent experiments using duplicates ( n = 4; values are means ± standard deviations [SD]). (C) Plaque phenotype in HuH7 cells of rMERS-CoV nsp14-E191D and wt control in the absence or presence of the mutagenic agent 5-FU. (D) Dose-response curve of wt and E191D mutant MERS-CoV in the presence of 5-FU concentrations up to 400 μM (MOI, 0.1; n = 4; means ± SD). Statistical significance of the difference with wt virus at each time point (A and B) or concentration (D) was assessed by paired t test. *, P

    Techniques Used: Infection, Plaque Assay, Mutagenesis, Concentration Assay

    40) Product Images from "Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH"

    Article Title: Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH

    Journal: Journal of Virology

    doi: 10.1128/JVI.02727-15

    Nucleolar accumulation of NS5 responds rapidly and reversibly in response to extracellular pH. (A) CLSM images of live Vero cells transfected to express GFP or DENV-2 GFP-NS5 and imaged
    Figure Legend Snippet: Nucleolar accumulation of NS5 responds rapidly and reversibly in response to extracellular pH. (A) CLSM images of live Vero cells transfected to express GFP or DENV-2 GFP-NS5 and imaged

    Techniques Used: Confocal Laser Scanning Microscopy, Transfection

    NS5 localizes within the nucleolus during DENV infection and in response to low pH. (A) CLSM images of DENV-2-infected Vero cells fixed and immunostained for endogenous nucleolar marker nucleolin and DENV-2 NS5 (top) or Vero cells cotransfected to express GFP-nucleolin and either mCherry or DENV-2 mCherry-NS5 (bottom) and imaged live. White arrows indicate nucleoli. The yellow dashed line indicates the region where the Plot Profile was performed to obtain the graphs in panel B. (B) The ImageJ plot profile tool was used to measure pixel intensity (fluorescence) along the line of interest (in panel A), with the resulting data plotted using Prism 6. (C) Vero cells transfected to express GFP-NS5 (DENV-2), treated with various stresses, including acidic pH (6.0), basic pH (8.5), 100 μM CoCl 2 (hypoxia), or hyper- or hypo-osmotic stress prior to live cell imaging. DIC images are shown to the right to identify the nucleoli (white and black arrows in GFP and DIC images, respectively). (D) Quantitative analysis of images such as those in panel C to determine the ratio of fluorescence intensity in the nucleolus (Fnu) compared with the nucleus (Fn); the “Fnu/n ratio.” The results represent the means + the standards error of the mean (SEM), where n ≥ 22. Statistical analysis by Student t test was performed using GraphPad Prism software (****, P
    Figure Legend Snippet: NS5 localizes within the nucleolus during DENV infection and in response to low pH. (A) CLSM images of DENV-2-infected Vero cells fixed and immunostained for endogenous nucleolar marker nucleolin and DENV-2 NS5 (top) or Vero cells cotransfected to express GFP-nucleolin and either mCherry or DENV-2 mCherry-NS5 (bottom) and imaged live. White arrows indicate nucleoli. The yellow dashed line indicates the region where the Plot Profile was performed to obtain the graphs in panel B. (B) The ImageJ plot profile tool was used to measure pixel intensity (fluorescence) along the line of interest (in panel A), with the resulting data plotted using Prism 6. (C) Vero cells transfected to express GFP-NS5 (DENV-2), treated with various stresses, including acidic pH (6.0), basic pH (8.5), 100 μM CoCl 2 (hypoxia), or hyper- or hypo-osmotic stress prior to live cell imaging. DIC images are shown to the right to identify the nucleoli (white and black arrows in GFP and DIC images, respectively). (D) Quantitative analysis of images such as those in panel C to determine the ratio of fluorescence intensity in the nucleolus (Fnu) compared with the nucleus (Fn); the “Fnu/n ratio.” The results represent the means + the standards error of the mean (SEM), where n ≥ 22. Statistical analysis by Student t test was performed using GraphPad Prism software (****, P

    Techniques Used: Infection, Confocal Laser Scanning Microscopy, Marker, Fluorescence, Transfection, Live Cell Imaging, Software

    Related Articles

    Infection:

    Article Title: Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes
    Article Snippet: The Vero cells and 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (16000-044, Gibco). .. For ZIKV infection, the Vero cells were maintained in VP-SFM medium (11681-020, Gibco). .. The Vero and 293T cell lines were purchased from the ATCC (CCL-81, CRL-3216).

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    Thermo Fisher vero cells
    Cytotoxicity of wild-type and mutant TcdB. <t>Vero</t> cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and <t>LDH</t> activity in supernatants was
    Vero Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ibv infected vero cells
    Regulation of Mcl-1 expression by MEK-1, PI3K and GADD153. (A) Induction of Mcl-1 in <t>IBV-infected</t> cells in the presence or absence of either 20 mM of MEK-1 inhibitor U0126 or 40 mM of PI3K inhibitor LY294002. <t>Vero,</t> and H1299 cells were incubated with normal medium (DMSO-), LY294002 in DMSO, U0129 in DMSO and DMSO only (DMSO+) for 1 hour, and then infected with IBV at a multiplicity of infection of approximately 2 in the presence or absence of the inhibitors. Cells were harvested at 16 hours post-infection, and total RNA extracted. The relative amounts of Mcl-1 transcripts were determined by quantitative RT-PCR and normalized against GAPDH. The relative fold of Mcl-1 induction in IBV-infected cells was determined by comparing with mock-infected cells. (B) Induction of Mcl-1 in IBV-infected cells by the pro-apoptotic transcription factor GADD153. H1299 cells were transfected with either siGADD153 or a non-targeting control for 72 hours and subsequently infected with IBV. Cells were harvested at 16, 18 and 20 hours post infection for western blot analysis using specific antibodies against the indicated proteins, with anti-actin as a loading control. M, mock infection.
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    Thermo Fisher vero cells with or without trypsin
    Trypsin-dependence and propagation of <t>PEDV</t> isolates 2013-A and NJ in <t>Vero/TMPRSS2,</t> Vero/MSPL cells and Vero cells (with or without trypsin). (a and b) Trypsin-dependence of 2013-A and NJ were determined by RT-PCR assay with the 2 −∆∆Ct method as described above. Error bars indicate the standard error of three independent experiments and the viral mRNA relative quantity of the Trypsin(-) group set to 1. (* p
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    Cytotoxicity of wild-type and mutant TcdB. Vero cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and LDH activity in supernatants was

    Journal: Infection and Immunity

    Article Title: Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

    doi: 10.1128/IAI.02316-14

    Figure Lengend Snippet: Cytotoxicity of wild-type and mutant TcdB. Vero cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and LDH activity in supernatants was

    Article Snippet: Cytotoxicity of Vero cells was measured by a lactate dehydrogenase (LDH) cytotoxicity kit (Pierce) according to the manufacturer's instructions.

    Techniques: Mutagenesis, Activity Assay

    Regulation of Mcl-1 expression by MEK-1, PI3K and GADD153. (A) Induction of Mcl-1 in IBV-infected cells in the presence or absence of either 20 mM of MEK-1 inhibitor U0126 or 40 mM of PI3K inhibitor LY294002. Vero, and H1299 cells were incubated with normal medium (DMSO-), LY294002 in DMSO, U0129 in DMSO and DMSO only (DMSO+) for 1 hour, and then infected with IBV at a multiplicity of infection of approximately 2 in the presence or absence of the inhibitors. Cells were harvested at 16 hours post-infection, and total RNA extracted. The relative amounts of Mcl-1 transcripts were determined by quantitative RT-PCR and normalized against GAPDH. The relative fold of Mcl-1 induction in IBV-infected cells was determined by comparing with mock-infected cells. (B) Induction of Mcl-1 in IBV-infected cells by the pro-apoptotic transcription factor GADD153. H1299 cells were transfected with either siGADD153 or a non-targeting control for 72 hours and subsequently infected with IBV. Cells were harvested at 16, 18 and 20 hours post infection for western blot analysis using specific antibodies against the indicated proteins, with anti-actin as a loading control. M, mock infection.

    Journal: PLoS ONE

    Article Title: Up-Regulation of Mcl-1 and Bak by Coronavirus Infection of Human, Avian and Animal Cells Modulates Apoptosis and Viral Replication

    doi: 10.1371/journal.pone.0030191

    Figure Lengend Snippet: Regulation of Mcl-1 expression by MEK-1, PI3K and GADD153. (A) Induction of Mcl-1 in IBV-infected cells in the presence or absence of either 20 mM of MEK-1 inhibitor U0126 or 40 mM of PI3K inhibitor LY294002. Vero, and H1299 cells were incubated with normal medium (DMSO-), LY294002 in DMSO, U0129 in DMSO and DMSO only (DMSO+) for 1 hour, and then infected with IBV at a multiplicity of infection of approximately 2 in the presence or absence of the inhibitors. Cells were harvested at 16 hours post-infection, and total RNA extracted. The relative amounts of Mcl-1 transcripts were determined by quantitative RT-PCR and normalized against GAPDH. The relative fold of Mcl-1 induction in IBV-infected cells was determined by comparing with mock-infected cells. (B) Induction of Mcl-1 in IBV-infected cells by the pro-apoptotic transcription factor GADD153. H1299 cells were transfected with either siGADD153 or a non-targeting control for 72 hours and subsequently infected with IBV. Cells were harvested at 16, 18 and 20 hours post infection for western blot analysis using specific antibodies against the indicated proteins, with anti-actin as a loading control. M, mock infection.

    Article Snippet: In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system.

    Techniques: Expressing, Infection, Incubation, Quantitative RT-PCR, Transfection, Western Blot

    Analysis of the expression of BCL2-related proteins in IBV-infected Vero cells, chicken fibroblast DF1 cells and chicken embryos. (A) Vero cells were infected with IBV, harvested at 0, 8, 16 and 24 hours post-infection, and lysates prepared. Western blot analysis was performed with the indicated specific antibodies, and probed with anti-actin as a loading control. (B) Chicken fibroblast DF1 cells were either infected with IBV, harvested at 8 and 16 hours post-infection and RNA extracted. RT-PCR analysis was carried out using specific primers for the indicated genes, with GAPDH as a loading control. (C) 10-day-old chicken embryos were inoculated with either mock virus (M) or IBV (1000 plaque-forming units per egg) in a 37°C incubator for 48 hours. Total RNA was extracted from homogenized tissues and used for RT-PCR using specific primers as above (B).

    Journal: PLoS ONE

    Article Title: Up-Regulation of Mcl-1 and Bak by Coronavirus Infection of Human, Avian and Animal Cells Modulates Apoptosis and Viral Replication

    doi: 10.1371/journal.pone.0030191

    Figure Lengend Snippet: Analysis of the expression of BCL2-related proteins in IBV-infected Vero cells, chicken fibroblast DF1 cells and chicken embryos. (A) Vero cells were infected with IBV, harvested at 0, 8, 16 and 24 hours post-infection, and lysates prepared. Western blot analysis was performed with the indicated specific antibodies, and probed with anti-actin as a loading control. (B) Chicken fibroblast DF1 cells were either infected with IBV, harvested at 8 and 16 hours post-infection and RNA extracted. RT-PCR analysis was carried out using specific primers for the indicated genes, with GAPDH as a loading control. (C) 10-day-old chicken embryos were inoculated with either mock virus (M) or IBV (1000 plaque-forming units per egg) in a 37°C incubator for 48 hours. Total RNA was extracted from homogenized tissues and used for RT-PCR using specific primers as above (B).

    Article Snippet: In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system.

    Techniques: Expressing, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Analysis of Bak and Mcl-1 expression at the mRNA and protein levels in IBV-infected mammalian cells. (A) Northern blot analysis of Bak and Mcl-1 expression at the mRNA level in IBV-infected mammalian cells. Vero, H1299 and Huh7 cells infected with (A) IBV or (B) UV-IBV were harvested at 0, 8, 12, 16 and 24 hours post-infection, respectively, and total RNA was extracted. Northern blot analysis was carried out with specific probes for Bak and Mcl-1. The same membrane was also probed with a GAPDH probe as a loading control. (B). Western blot analysis of Bak and Mcl-1 expression at the protein level in mammalian cells. Vero, H1299 and Huh7 cells infected with IBV were harvested at 0, 8, 12, 16, 24 and 36 hours post-infection, respectively, and cell lysates prepared. Western blot analysis was performed using specific antibodies as indicated, with anti-actin as a loading control. M, mock infection.

    Journal: PLoS ONE

    Article Title: Up-Regulation of Mcl-1 and Bak by Coronavirus Infection of Human, Avian and Animal Cells Modulates Apoptosis and Viral Replication

    doi: 10.1371/journal.pone.0030191

    Figure Lengend Snippet: Analysis of Bak and Mcl-1 expression at the mRNA and protein levels in IBV-infected mammalian cells. (A) Northern blot analysis of Bak and Mcl-1 expression at the mRNA level in IBV-infected mammalian cells. Vero, H1299 and Huh7 cells infected with (A) IBV or (B) UV-IBV were harvested at 0, 8, 12, 16 and 24 hours post-infection, respectively, and total RNA was extracted. Northern blot analysis was carried out with specific probes for Bak and Mcl-1. The same membrane was also probed with a GAPDH probe as a loading control. (B). Western blot analysis of Bak and Mcl-1 expression at the protein level in mammalian cells. Vero, H1299 and Huh7 cells infected with IBV were harvested at 0, 8, 12, 16, 24 and 36 hours post-infection, respectively, and cell lysates prepared. Western blot analysis was performed using specific antibodies as indicated, with anti-actin as a loading control. M, mock infection.

    Article Snippet: In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system.

    Techniques: Expressing, Infection, Northern Blot, Western Blot

    Trypsin-dependence and propagation of PEDV isolates 2013-A and NJ in Vero/TMPRSS2, Vero/MSPL cells and Vero cells (with or without trypsin). (a and b) Trypsin-dependence of 2013-A and NJ were determined by RT-PCR assay with the 2 −∆∆Ct method as described above. Error bars indicate the standard error of three independent experiments and the viral mRNA relative quantity of the Trypsin(-) group set to 1. (* p

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: Trypsin-dependence and propagation of PEDV isolates 2013-A and NJ in Vero/TMPRSS2, Vero/MSPL cells and Vero cells (with or without trypsin). (a and b) Trypsin-dependence of 2013-A and NJ were determined by RT-PCR assay with the 2 −∆∆Ct method as described above. Error bars indicate the standard error of three independent experiments and the viral mRNA relative quantity of the Trypsin(-) group set to 1. (* p

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    The propagation of cell-adapted PEDV strain LJB/03 P23 in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL cells. Ultrathin sections of PEDV LJB/03-infected Vero cells at 24 h post-infection were prepared, and massive virus particles as shown by the arrow (Bar = 200 nm) were observed by the electron microscopy (a); PEDV LJB/03 particles in culture media as shown by the arrow (Bar = 200 nm) were observed by the transmission electron microscopy (b). (c) Trypsin-dependence of LJB/03 P23 was determined by RT-PCR assay with SYBR Premix EX Taq II (* p

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: The propagation of cell-adapted PEDV strain LJB/03 P23 in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL cells. Ultrathin sections of PEDV LJB/03-infected Vero cells at 24 h post-infection were prepared, and massive virus particles as shown by the arrow (Bar = 200 nm) were observed by the electron microscopy (a); PEDV LJB/03 particles in culture media as shown by the arrow (Bar = 200 nm) were observed by the transmission electron microscopy (b). (c) Trypsin-dependence of LJB/03 P23 was determined by RT-PCR assay with SYBR Premix EX Taq II (* p

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Infection, Electron Microscopy, Transmission Assay, Reverse Transcription Polymerase Chain Reaction

    The cytopathic effects (CPEs) of Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) incubated with PEDV isolates 2013-A and NJ. The Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) were, respectively, incubated with PEDV isolates (2013-A and NJ) at an MOI = 0.01, followed by observation of CPEs. (a) CPEs observation of trypsin-dependent PEDV 2013-A at 24, 48, and 72 h post-infection; (b) CPEs observation of trypsin-dependent PEDV NJ at 24, 48, and 72 h post-infection. Arrows indicate syncytium.

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: The cytopathic effects (CPEs) of Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) incubated with PEDV isolates 2013-A and NJ. The Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) were, respectively, incubated with PEDV isolates (2013-A and NJ) at an MOI = 0.01, followed by observation of CPEs. (a) CPEs observation of trypsin-dependent PEDV 2013-A at 24, 48, and 72 h post-infection; (b) CPEs observation of trypsin-dependent PEDV NJ at 24, 48, and 72 h post-infection. Arrows indicate syncytium.

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Incubation, Infection

    A schematic diagram for the construction of Vero/TMPRSS2 and Vero/MSPL cell lines, which were generated following the steps of arrows. (a) The constitutive recombinant plasmids (lentiviral vector) FUGW-TMPRSS2 and FUGW-MSPL containing TMPRSS2 and Vero/MSPL genes, (b) lentivirus envelope plasmids pMD2.G and psPAX2, (c) pseudotyped lentivirus particles produced in the HEK293 T cells, transduced to the Vero cells, and established in the cell lines by screening of the fluorescence expression.

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: A schematic diagram for the construction of Vero/TMPRSS2 and Vero/MSPL cell lines, which were generated following the steps of arrows. (a) The constitutive recombinant plasmids (lentiviral vector) FUGW-TMPRSS2 and FUGW-MSPL containing TMPRSS2 and Vero/MSPL genes, (b) lentivirus envelope plasmids pMD2.G and psPAX2, (c) pseudotyped lentivirus particles produced in the HEK293 T cells, transduced to the Vero cells, and established in the cell lines by screening of the fluorescence expression.

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Generated, Recombinant, Plasmid Preparation, Produced, Fluorescence, Expressing

    Stable expression of TMPRSS2 and MSPL genes in Vero cell lines. (a) The proteins in the lysates of Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages were separated by SDS-PAGE, followed by identification by western blot with anti-HA monoclonal antibody. FUGW: FUGW/Vero cell lines; TMPRSS2: Vero/TMPRSS2 cell lines; MSPL: Vero/MSPL cell lines. (b) TMPRSS2 and MSPL genes were detected in Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages by reverse transcription PCR. M: DNA Marker; N: negative control (water as negative control); FUGW: FUGW/Vero cell line. (c) Fluorescence images of Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages were acquired by the fluorescence microscope, using FUGW/Vero cells as control. (d) Fluorescence intensity of Vero/TMPRSS2. Vero/MSPL and Vero cells were detected by flow cytometry after 15 serial passages.

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: Stable expression of TMPRSS2 and MSPL genes in Vero cell lines. (a) The proteins in the lysates of Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages were separated by SDS-PAGE, followed by identification by western blot with anti-HA monoclonal antibody. FUGW: FUGW/Vero cell lines; TMPRSS2: Vero/TMPRSS2 cell lines; MSPL: Vero/MSPL cell lines. (b) TMPRSS2 and MSPL genes were detected in Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages by reverse transcription PCR. M: DNA Marker; N: negative control (water as negative control); FUGW: FUGW/Vero cell line. (c) Fluorescence images of Vero/TMPRSS2 and Vero/MSPL cell lines after consecutively incubating 15 passages were acquired by the fluorescence microscope, using FUGW/Vero cells as control. (d) Fluorescence intensity of Vero/TMPRSS2. Vero/MSPL and Vero cells were detected by flow cytometry after 15 serial passages.

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Expressing, SDS Page, Western Blot, Polymerase Chain Reaction, Marker, Negative Control, Fluorescence, Microscopy, Flow Cytometry

    Immunofluorescence of Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) infected by PEDV isolates 2013-A and NJ at an MOI = 0.01. (a) The fluorescence intensity was observed in trypsin-dependent PEDV 2013-A at 48 h post-infection. (b) The fluorescence intensity of trypsin-dependent PEDV NJ at 48 h post-infection.

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: Immunofluorescence of Vero/TMPRSS2, Vero/MSPL, and Vero cells (without or with 3 μg/mL trypsin) infected by PEDV isolates 2013-A and NJ at an MOI = 0.01. (a) The fluorescence intensity was observed in trypsin-dependent PEDV 2013-A at 48 h post-infection. (b) The fluorescence intensity of trypsin-dependent PEDV NJ at 48 h post-infection.

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Immunofluorescence, Infection, Fluorescence

    Proteolytic activation of PEDV S protein in Vero, Vero/TMPRSS2 and Vero/MSPL cells. (a) The expression of PEDV S genes in the Vero cells. Vero cells were transfected with PEDV-S plasmids (pCMV-HA-S encoding PEDV LJB/03 S protein with a HA tag) and the expression of PEDV S genes in the Vero cells were detected by western blot with anti-HA monoclonal antibody after 72 h post-transfection. Empty pCMV-HA plasmid as control. (b and c) Cleavage of PEDV S protein in Vero cells (with or with trypsin), Vero/TMPRSS2 and Vero/MSPL cells. Arrows indicate either uncleaved S protein (black arrows) or N-terminal cleavage S protein products (white arrow). Empty pCMV-HA plasmid as control.

    Journal: Virulence

    Article Title: Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

    doi: 10.1080/21505594.2020.1770491

    Figure Lengend Snippet: Proteolytic activation of PEDV S protein in Vero, Vero/TMPRSS2 and Vero/MSPL cells. (a) The expression of PEDV S genes in the Vero cells. Vero cells were transfected with PEDV-S plasmids (pCMV-HA-S encoding PEDV LJB/03 S protein with a HA tag) and the expression of PEDV S genes in the Vero cells were detected by western blot with anti-HA monoclonal antibody after 72 h post-transfection. Empty pCMV-HA plasmid as control. (b and c) Cleavage of PEDV S protein in Vero cells (with or with trypsin), Vero/TMPRSS2 and Vero/MSPL cells. Arrows indicate either uncleaved S protein (black arrows) or N-terminal cleavage S protein products (white arrow). Empty pCMV-HA plasmid as control.

    Article Snippet: The replication kinetics of intracellular PEDV LJB/03 P23 RNA in Vero cells (with or without trypsin), Vero/TMPRSS2 and Vero/MSPL stable cell lines were determined by RT-PCR assay.

    Techniques: Activation Assay, Expressing, Transfection, Western Blot, Plasmid Preparation