vero cells  (Millipore)


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    Name:
    Vero cells
    Description:
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
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    v0180000
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    Structured Review

    Millipore vero cells
    Induction of type I IFN expression in monocytes requires virus infection. (A-B) Enriched WT bone marrow monocytes were co-cultured with <t>Vero</t> cells infected with RRV-GFP (n = 3/group). After 18 h of co-culture, GFP expression in Vero cells and monocytes was measured by flow cytometry. (A) Shown are representative histograms. (B) The percent GFP + monocytes and Vero cells. Data are representative of two independent experiments. (C-D) Enriched WT bone marrow monocytes were co-cultured with uninfected or RRV-GFP-infected Vero cells in the presence or absence of 1 μM <t>Latrunculin</t> B. After 18 h of co-culture, (C) IFNα2 mRNA expression level in monocytes was quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression ( n -fold increase) over expression in uninfected Vero cells without monocytes. (D) The percent GFP + Vero cells and monocytes were measured by flow cytometry based on GFP expression within the CD45 - Vero cells and the CD45 + monocytes. Data are combined from two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test.
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
    https://www.bioz.com/result/vero cells/product/Millipore
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    vero cells - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Inflammatory monocytes mediate control of acute alphavirus infection in mice"

    Article Title: Inflammatory monocytes mediate control of acute alphavirus infection in mice

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006748

    Induction of type I IFN expression in monocytes requires virus infection. (A-B) Enriched WT bone marrow monocytes were co-cultured with Vero cells infected with RRV-GFP (n = 3/group). After 18 h of co-culture, GFP expression in Vero cells and monocytes was measured by flow cytometry. (A) Shown are representative histograms. (B) The percent GFP + monocytes and Vero cells. Data are representative of two independent experiments. (C-D) Enriched WT bone marrow monocytes were co-cultured with uninfected or RRV-GFP-infected Vero cells in the presence or absence of 1 μM Latrunculin B. After 18 h of co-culture, (C) IFNα2 mRNA expression level in monocytes was quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression ( n -fold increase) over expression in uninfected Vero cells without monocytes. (D) The percent GFP + Vero cells and monocytes were measured by flow cytometry based on GFP expression within the CD45 - Vero cells and the CD45 + monocytes. Data are combined from two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test.
    Figure Legend Snippet: Induction of type I IFN expression in monocytes requires virus infection. (A-B) Enriched WT bone marrow monocytes were co-cultured with Vero cells infected with RRV-GFP (n = 3/group). After 18 h of co-culture, GFP expression in Vero cells and monocytes was measured by flow cytometry. (A) Shown are representative histograms. (B) The percent GFP + monocytes and Vero cells. Data are representative of two independent experiments. (C-D) Enriched WT bone marrow monocytes were co-cultured with uninfected or RRV-GFP-infected Vero cells in the presence or absence of 1 μM Latrunculin B. After 18 h of co-culture, (C) IFNα2 mRNA expression level in monocytes was quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression ( n -fold increase) over expression in uninfected Vero cells without monocytes. (D) The percent GFP + Vero cells and monocytes were measured by flow cytometry based on GFP expression within the CD45 - Vero cells and the CD45 + monocytes. Data are combined from two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test.

    Techniques Used: Expressing, Infection, Cell Culture, Co-Culture Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Over Expression

    2) Product Images from "Evaluation of the Antiviral Potential of Halogenated Dihydrorugosaflavonoids and Molecular Modeling with nsP3 Protein of Chikungunya Virus (CHIKV)"

    Article Title: Evaluation of the Antiviral Potential of Halogenated Dihydrorugosaflavonoids and Molecular Modeling with nsP3 Protein of Chikungunya Virus (CHIKV)

    Journal: ACS Omega

    doi: 10.1021/acsomega.9b02900

    Demonstration of antiviral activity against CHIKV in treated Vero cells. Dihydrorugosaflavonoid-treated Vero cells (pretreated cells) were infected with CHIKV at an MOI of 1 for 90 min. Followed by virus adsorption, the infection medium was changed with the medium containing compounds for 24 h. After 24 hpi, the supernatant was collected for determining percent reduction of viral titer by plaque reduction assay: (a) 5c compound; (b) 5d compound. The graphs show the dose-dependent curves of 5c and 5d . In the analysis, EC 50 values of 5c and 5d are 26.14 and 28.39 μM, respectively. EC 50 thresholds are marked with a dotted line. Statistical significance is analyzed by nonlinear regression (curve fit). Values are mean ± standard deviation of two experiments, which are performed in triplicates.
    Figure Legend Snippet: Demonstration of antiviral activity against CHIKV in treated Vero cells. Dihydrorugosaflavonoid-treated Vero cells (pretreated cells) were infected with CHIKV at an MOI of 1 for 90 min. Followed by virus adsorption, the infection medium was changed with the medium containing compounds for 24 h. After 24 hpi, the supernatant was collected for determining percent reduction of viral titer by plaque reduction assay: (a) 5c compound; (b) 5d compound. The graphs show the dose-dependent curves of 5c and 5d . In the analysis, EC 50 values of 5c and 5d are 26.14 and 28.39 μM, respectively. EC 50 thresholds are marked with a dotted line. Statistical significance is analyzed by nonlinear regression (curve fit). Values are mean ± standard deviation of two experiments, which are performed in triplicates.

    Techniques Used: Activity Assay, Infection, Adsorption, Standard Deviation

    Dihydrorugosaflavonoid cytotoxicity and inhibition assay. (a and b) Cell survival dose–response curves to determine the cytotoxicity of dihydrorugosaflavonoid compounds in Vero cell lines. Compounds’ treatment at different concentrations, 10–100 μM, was given to Vero cells for 24 h. (a) 5c- compound-treated Vero cells; (b) 5d -compound-treated Vero cells. Percent survival values were obtained based on untreated cells as control. CC 50 thresholds are marked with a dotted line; (c and d) dose-dependent inhibition of CHIKV shown by 5c and 5d , respectively. 5c and 5d both exhibit a statistically significant reduction in CHIKV titer at micromolar concentrations. In the analysis, statistical significance is analyzed by the one-way analysis of variance (ANOVA) test and Dunnett’s post-test. *** P
    Figure Legend Snippet: Dihydrorugosaflavonoid cytotoxicity and inhibition assay. (a and b) Cell survival dose–response curves to determine the cytotoxicity of dihydrorugosaflavonoid compounds in Vero cell lines. Compounds’ treatment at different concentrations, 10–100 μM, was given to Vero cells for 24 h. (a) 5c- compound-treated Vero cells; (b) 5d -compound-treated Vero cells. Percent survival values were obtained based on untreated cells as control. CC 50 thresholds are marked with a dotted line; (c and d) dose-dependent inhibition of CHIKV shown by 5c and 5d , respectively. 5c and 5d both exhibit a statistically significant reduction in CHIKV titer at micromolar concentrations. In the analysis, statistical significance is analyzed by the one-way analysis of variance (ANOVA) test and Dunnett’s post-test. *** P

    Techniques Used: Inhibition

    Evaluation of the antiviral effect by indirect immunofluorescence assay (IFA). Viral inhibition was assessed by IFA by treatment with 5c and 5d compounds. Cells were observed at 36 hpi; micrographs with green fluorescence indicate the virus load as determined with anti-E2 mAb and fluorescein isothiocyanate (FITC) conjugated secondary antibody (green), and blue fluorescence indicates the nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Infected Vero cells with compound treatment ( 5c , 70 μM (c); 5c , 50 μM (d); 5d , 70 μM (e); 5d , 50 μM (f)) and cell control (containing no virus (a)) are shown. Virus control images are represented in (b). Cells were observed by a fluorescent microscope (Leica Microsystems) with a 20× objective lens. Scale bar is 50 μm.
    Figure Legend Snippet: Evaluation of the antiviral effect by indirect immunofluorescence assay (IFA). Viral inhibition was assessed by IFA by treatment with 5c and 5d compounds. Cells were observed at 36 hpi; micrographs with green fluorescence indicate the virus load as determined with anti-E2 mAb and fluorescein isothiocyanate (FITC) conjugated secondary antibody (green), and blue fluorescence indicates the nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Infected Vero cells with compound treatment ( 5c , 70 μM (c); 5c , 50 μM (d); 5d , 70 μM (e); 5d , 50 μM (f)) and cell control (containing no virus (a)) are shown. Virus control images are represented in (b). Cells were observed by a fluorescent microscope (Leica Microsystems) with a 20× objective lens. Scale bar is 50 μm.

    Techniques Used: Immunofluorescence, Inhibition, Fluorescence, Staining, Infection, Microscopy

    (a, b) Relative quantification of CHIKV mRNA of Vero cells treated with 5c and 5d compounds, by real-time PCR. Compounds were added to the cells 12 h before CHIKV infection and were present in the post-infection medium. Viral RNA in infected cells was quantified 24 h post-infection using β-actin as endogenous control by qRT-PCR performed in triplicates. By using the comparative Ct method, the relative amount of mRNA was calculated. The fold change in treated cells was compared with virus control and is presented logarithmically. Statistical significance is analyzed by the one-way ANOVA test and Dunnett’s post-test. *** P
    Figure Legend Snippet: (a, b) Relative quantification of CHIKV mRNA of Vero cells treated with 5c and 5d compounds, by real-time PCR. Compounds were added to the cells 12 h before CHIKV infection and were present in the post-infection medium. Viral RNA in infected cells was quantified 24 h post-infection using β-actin as endogenous control by qRT-PCR performed in triplicates. By using the comparative Ct method, the relative amount of mRNA was calculated. The fold change in treated cells was compared with virus control and is presented logarithmically. Statistical significance is analyzed by the one-way ANOVA test and Dunnett’s post-test. *** P

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Quantitative RT-PCR

    3) Product Images from "Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, which Demonstrates Attenuation and Immunogenicity in Mice"

    Article Title: Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, which Demonstrates Attenuation and Immunogenicity in Mice

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.14-0427

    Sensitivity of Japanese encephalitis virus (JEV) strains to type I interferon (IFN)-treatment of Vero cells. Vero cells were infected at an multiplicity of infection (MOI) of 0.01 PFU/cell, followed by addition of medium-only (untreated) or medium containing
    Figure Legend Snippet: Sensitivity of Japanese encephalitis virus (JEV) strains to type I interferon (IFN)-treatment of Vero cells. Vero cells were infected at an multiplicity of infection (MOI) of 0.01 PFU/cell, followed by addition of medium-only (untreated) or medium containing

    Techniques Used: Infection

    4) Product Images from "Trypanocidal Activity of Smallanthus sonchifolius: Identification of Active Sesquiterpene Lactones by Bioassay-Guided Fractionation"

    Article Title: Trypanocidal Activity of Smallanthus sonchifolius: Identification of Active Sesquiterpene Lactones by Bioassay-Guided Fractionation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/627898

    Cytotoxicity of enhydrin, uvedalin, and polymatin B on Vero cells. Cultures were kept for 24 h in the presence of different concentrations (1 to 50 μ g/mL) of the STLs. Cell viability was determined by the MTT method and was expressed as the ratio between viable cells in the presence and absence of the compound multiplied by 100. Bars represent the mean ± SEM of three experiments carried out in duplicate.
    Figure Legend Snippet: Cytotoxicity of enhydrin, uvedalin, and polymatin B on Vero cells. Cultures were kept for 24 h in the presence of different concentrations (1 to 50 μ g/mL) of the STLs. Cell viability was determined by the MTT method and was expressed as the ratio between viable cells in the presence and absence of the compound multiplied by 100. Bars represent the mean ± SEM of three experiments carried out in duplicate.

    Techniques Used: MTT Assay

    5) Product Images from "Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic"

    Article Title: Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic

    Journal: Journal of Virology

    doi: 10.1128/JVI.02615-15

    Schematic representation of the genome of rMACV/Cd#1-GPC and the virus growth curves. (A) For rMACV/Cd#1-GPC, the entire MACV GPC gene was replaced with the Cd#1 GPC gene. The plaque morphology is shown. (B) The growth of rMACV/Cd#1-GPC was characterized in Vero cells (MOI = 0.01). The titer of rMACV was significantly higher than that of rCd#1 at 48 hpi ( n = 5; **, P
    Figure Legend Snippet: Schematic representation of the genome of rMACV/Cd#1-GPC and the virus growth curves. (A) For rMACV/Cd#1-GPC, the entire MACV GPC gene was replaced with the Cd#1 GPC gene. The plaque morphology is shown. (B) The growth of rMACV/Cd#1-GPC was characterized in Vero cells (MOI = 0.01). The titer of rMACV was significantly higher than that of rCd#1 at 48 hpi ( n = 5; **, P

    Techniques Used: Gel Permeation Chromatography

    6) Product Images from "Ras-GAP Binding and Phosphorylation by Herpes Simplex Virus Type 2 RR1 PK (ICP10) and Activation of the Ras/MEK/MAPK Mitogenic Pathway Are Required for Timely Onset of Virus Growth"

    Article Title: Ras-GAP Binding and Phosphorylation by Herpes Simplex Virus Type 2 RR1 PK (ICP10) and Activation of the Ras/MEK/MAPK Mitogenic Pathway Are Required for Timely Onset of Virus Growth

    Journal: Journal of Virology

    doi:

    Onset of HSV-2 growth is delayed by PD98059. Vero cells were pretreated (1 h, 37°C) with 50 μM PD98059 (○) or PD98059 reconstitution medium (●) and infected with HSV-2. They were reincubated in medium containing 1% FCS with or without PD98059, respectively. Adsorption was for 1 h at 37°C (0 h of growth curve). Virus titers were determined at 2 to 48 h after adsorption, and the results are expressed as the burst size (PFU/cell ± the standard error of the mean). Cells infected with ICP10ΔPK in medium containing 10% FCS (▵) were studied in parallel.
    Figure Legend Snippet: Onset of HSV-2 growth is delayed by PD98059. Vero cells were pretreated (1 h, 37°C) with 50 μM PD98059 (○) or PD98059 reconstitution medium (●) and infected with HSV-2. They were reincubated in medium containing 1% FCS with or without PD98059, respectively. Adsorption was for 1 h at 37°C (0 h of growth curve). Virus titers were determined at 2 to 48 h after adsorption, and the results are expressed as the burst size (PFU/cell ± the standard error of the mean). Cells infected with ICP10ΔPK in medium containing 10% FCS (▵) were studied in parallel.

    Techniques Used: Infection, Adsorption

    7) Product Images from "A 176 amino acid polypeptide derived from the mumps virus HN ectodomain shows immunological and biological properties similar to the HN protein"

    Article Title: A 176 amino acid polypeptide derived from the mumps virus HN ectodomain shows immunological and biological properties similar to the HN protein

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-195

    Expression of the pcDNAHN176-construct . A) Detection of HN176 mRNA in transfected Vero cells by RT-PCR amplification. Lane 1) ϕX174 DNA- HaeIII marker; Lane 2) RT/PCR negative control; Lane 3) Positive control (MuV-infected cells); Lane 4) Vero-untransfected cells, 5) Vero cells transfected with the plasmid pcDNA3.1, Lane 6) Vero cells transfected with the pcDNA-HN176-construct. The arrow indicates 580 bp amplicon. 1% agarose gel/100 V/1 hr/49 mA. B ) RT-PCR Controls. Lane 1) ϕX174 DNA- HaeIII marker; Lane 2) PCR amplification of the RNA samples obtained from pcDNAHN176-transfected Vero cells without a previous RT reaction; Lane 3 and 4) RT/PCR amplification of β-actin gene using RNA samples obtained from HeLa cells and pcDNAHN176-transfected Vero cells, respectively. C ) Immunodetection of the HN176 polypeptide by immunochemistry (1 st row) and immunofluorescence (2 nd row). Frames 1 5 mock infected cells; 2 6 pcDNA3.1-transfected cells; 3 7 MuV-infected cells; 4 8 pcDNAHN176-transfected cells, the blue (immunochemistry) and the green (immunofluorescence) colors indicate a positive reaction, N indicates the nucleus. 40×
    Figure Legend Snippet: Expression of the pcDNAHN176-construct . A) Detection of HN176 mRNA in transfected Vero cells by RT-PCR amplification. Lane 1) ϕX174 DNA- HaeIII marker; Lane 2) RT/PCR negative control; Lane 3) Positive control (MuV-infected cells); Lane 4) Vero-untransfected cells, 5) Vero cells transfected with the plasmid pcDNA3.1, Lane 6) Vero cells transfected with the pcDNA-HN176-construct. The arrow indicates 580 bp amplicon. 1% agarose gel/100 V/1 hr/49 mA. B ) RT-PCR Controls. Lane 1) ϕX174 DNA- HaeIII marker; Lane 2) PCR amplification of the RNA samples obtained from pcDNAHN176-transfected Vero cells without a previous RT reaction; Lane 3 and 4) RT/PCR amplification of β-actin gene using RNA samples obtained from HeLa cells and pcDNAHN176-transfected Vero cells, respectively. C ) Immunodetection of the HN176 polypeptide by immunochemistry (1 st row) and immunofluorescence (2 nd row). Frames 1 5 mock infected cells; 2 6 pcDNA3.1-transfected cells; 3 7 MuV-infected cells; 4 8 pcDNAHN176-transfected cells, the blue (immunochemistry) and the green (immunofluorescence) colors indicate a positive reaction, N indicates the nucleus. 40×

    Techniques Used: Expressing, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Negative Control, Positive Control, Infection, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Immunodetection, Immunofluorescence

    Hemadsorption and neuraminidase activities in the pcDNAHN176 transfected cells . A) . Hemadsorption (HD) reaction. a) Mock infected cells; b) MuV-infected cells; c) pcDNA-3.1-transfected cells; d) pcDNAHN176-transfected Vero cells. The red cell aggregates indicate a positive HD. 40×. B ) Neuraminidase (NA) reaction in total cellular protein extract by a dot blot assay: a) Mock infected cells; b) MuV-infected cells; c) pcDNA-3.1-transfected cells; d) pcDNAHN176-transfected Vero cells. C) % of hemoglobin (Hb) released after the HD. The Hb absorbance of MuV-infected cells was considered as 100% and was used to calculate the % of Hb for the different cells. D ) Comparison of the NA activity in MuV-infected cells, pcDNA3.1-transfected cells and pcDNAHN176-transfected Vero cells by dot blot and spectrophotometric methods.
    Figure Legend Snippet: Hemadsorption and neuraminidase activities in the pcDNAHN176 transfected cells . A) . Hemadsorption (HD) reaction. a) Mock infected cells; b) MuV-infected cells; c) pcDNA-3.1-transfected cells; d) pcDNAHN176-transfected Vero cells. The red cell aggregates indicate a positive HD. 40×. B ) Neuraminidase (NA) reaction in total cellular protein extract by a dot blot assay: a) Mock infected cells; b) MuV-infected cells; c) pcDNA-3.1-transfected cells; d) pcDNAHN176-transfected Vero cells. C) % of hemoglobin (Hb) released after the HD. The Hb absorbance of MuV-infected cells was considered as 100% and was used to calculate the % of Hb for the different cells. D ) Comparison of the NA activity in MuV-infected cells, pcDNA3.1-transfected cells and pcDNAHN176-transfected Vero cells by dot blot and spectrophotometric methods.

    Techniques Used: Transfection, Infection, Dot Blot, Activity Assay

    Immunological properties of the pcDNAHN176-construct . A) Detection of the HN176 polypeptide and HN protein by Western Blot. Lane 1) Negative control, uninfected cells and sera from pcDNAHN176-immunized rabbits; Lane 2) MuV-infected-Vero cells and anti-MuV serum; Lane 3) pcDNAHN176-transfected cells and anti-MuV serum; Lane 4) MuV-infected-Vero cells and sera from pcDNAHN176-immunized rabbits; Lane 5) pcDNAHN176-transfected cells and sera from pcDNAHN176-immunized rabbits. The upper arrow indicates in lane 2 and 4 the position of the complete viral HN protein, and the lower arrow indicates the position of the HN176 polypeptide in lanes 3 5. B) Body gain weight of hamsters immunized and challenged with MuV. Group A, animals without immunization and uninfected; Group B, viral control group (animals without immunization and challenged with MuV); Group C, animals immunized with pcDNA3.1 without challenge; Group D, animals immunized with pcDNAHN176-construct without challenge; Group E, animals immunized with pcDNA3.1 and challenged with MuV; Group F, animals immunized with pcDNAHN176 and challenged with MuV. C) Virus isolation from different organs of the hamsters groups. MuV was detected by HD, quantifying the amount of Hb. D) Detection of IL associated to Th1 response in the hamsters groups. E) Detection of IL associated to Th2 response in the hamsters groups. ILs were measured using the Luminex System (Invitrogen ® ). F) Lymphoproliferation index of spleen cells obtained from the hamsters groups. Cell proliferation was measured by MTT method.
    Figure Legend Snippet: Immunological properties of the pcDNAHN176-construct . A) Detection of the HN176 polypeptide and HN protein by Western Blot. Lane 1) Negative control, uninfected cells and sera from pcDNAHN176-immunized rabbits; Lane 2) MuV-infected-Vero cells and anti-MuV serum; Lane 3) pcDNAHN176-transfected cells and anti-MuV serum; Lane 4) MuV-infected-Vero cells and sera from pcDNAHN176-immunized rabbits; Lane 5) pcDNAHN176-transfected cells and sera from pcDNAHN176-immunized rabbits. The upper arrow indicates in lane 2 and 4 the position of the complete viral HN protein, and the lower arrow indicates the position of the HN176 polypeptide in lanes 3 5. B) Body gain weight of hamsters immunized and challenged with MuV. Group A, animals without immunization and uninfected; Group B, viral control group (animals without immunization and challenged with MuV); Group C, animals immunized with pcDNA3.1 without challenge; Group D, animals immunized with pcDNAHN176-construct without challenge; Group E, animals immunized with pcDNA3.1 and challenged with MuV; Group F, animals immunized with pcDNAHN176 and challenged with MuV. C) Virus isolation from different organs of the hamsters groups. MuV was detected by HD, quantifying the amount of Hb. D) Detection of IL associated to Th1 response in the hamsters groups. E) Detection of IL associated to Th2 response in the hamsters groups. ILs were measured using the Luminex System (Invitrogen ® ). F) Lymphoproliferation index of spleen cells obtained from the hamsters groups. Cell proliferation was measured by MTT method.

    Techniques Used: Construct, Western Blot, Negative Control, Infection, Transfection, Virus Isolation Assay, Luminex, MTT Assay

    8) Product Images from "The potential molecular effects of bursal septpeptide II on immune induction and antitumor activity"

    Article Title: The potential molecular effects of bursal septpeptide II on immune induction and antitumor activity

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2015.16.3.325

    p53 transcription and protein expression after BSP-II treatment. Vero cells were transfected with p53 Luc and pRL-TK plasmid, and then cultured with or without BSP-II for 24 h. p53-luciferase activity levels were then measured (A). The transfected Vero cells were also pre-incubated with α-pifithrin for 2 h, incubated with or without BSP-II (2 µg/mL) for 22 h, and the level of p53-luciferase activity was measured (B). Non-transfected Vero cells were cultured with or without BSP-II for 24 h, and Western blotting analysis was performed to detect p53 and Bax protein expressions (C). Transfected or non-transfected Vero cells were treated with Dox as a positive control. Bars represent the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: p53 transcription and protein expression after BSP-II treatment. Vero cells were transfected with p53 Luc and pRL-TK plasmid, and then cultured with or without BSP-II for 24 h. p53-luciferase activity levels were then measured (A). The transfected Vero cells were also pre-incubated with α-pifithrin for 2 h, incubated with or without BSP-II (2 µg/mL) for 22 h, and the level of p53-luciferase activity was measured (B). Non-transfected Vero cells were cultured with or without BSP-II for 24 h, and Western blotting analysis was performed to detect p53 and Bax protein expressions (C). Transfected or non-transfected Vero cells were treated with Dox as a positive control. Bars represent the mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay, Incubation, Western Blot, Positive Control

    9) Product Images from "The Influenza Virus Protein PB1-F2 Interacts with IKK? and Modulates NF-?B Signalling"

    Article Title: The Influenza Virus Protein PB1-F2 Interacts with IKK? and Modulates NF-?B Signalling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063852

    PB1-F2 has sequence similarity with IKKα and interacts with IKKβ. A) Alignment of PB1-F2 Q/HK97 protein sequence with human and chicken IKKα using ClustalW (*denotes identical residues, : denotes conservative substitutions and. denotes semi-conservative substitutions). The grey shading denotes the conserved residues in PB1-F2 from PR8, T/Eng and T/Tur. B) Yeast cells were independently transformed with different combinations of pGADT7 (AD) and pGBKT7 (BD) constructs and plated on selective medium. Yeast cells co-transformed with BD PB1-F2 and AD PB1-F2 were used as a positive control for protein-protein interaction. C) Vero cells were transfected with the indicated constructs and forty eight hours after transfection, protein complexes were immunoprecipitated from cellular lysates using a monoclonal anti-myc antibody. Proteins in the immunoprecipitates and in the whole cell lysates (4% input) were evaluated by Western blot using anti-Flag and anti-myc antibodies. D) Vero cells were transfected with 300 ng of the different PB1-F2 expressing vectors and 300 ng of pcDNA3-IKKβ. The cellular localisation of the proteins was analyzed by confocal microscopy using anti-myc (PB1-F2, red) and anti-Flag (IKKβ, green) antibodies.
    Figure Legend Snippet: PB1-F2 has sequence similarity with IKKα and interacts with IKKβ. A) Alignment of PB1-F2 Q/HK97 protein sequence with human and chicken IKKα using ClustalW (*denotes identical residues, : denotes conservative substitutions and. denotes semi-conservative substitutions). The grey shading denotes the conserved residues in PB1-F2 from PR8, T/Eng and T/Tur. B) Yeast cells were independently transformed with different combinations of pGADT7 (AD) and pGBKT7 (BD) constructs and plated on selective medium. Yeast cells co-transformed with BD PB1-F2 and AD PB1-F2 were used as a positive control for protein-protein interaction. C) Vero cells were transfected with the indicated constructs and forty eight hours after transfection, protein complexes were immunoprecipitated from cellular lysates using a monoclonal anti-myc antibody. Proteins in the immunoprecipitates and in the whole cell lysates (4% input) were evaluated by Western blot using anti-Flag and anti-myc antibodies. D) Vero cells were transfected with 300 ng of the different PB1-F2 expressing vectors and 300 ng of pcDNA3-IKKβ. The cellular localisation of the proteins was analyzed by confocal microscopy using anti-myc (PB1-F2, red) and anti-Flag (IKKβ, green) antibodies.

    Techniques Used: Sequencing, Transformation Assay, Construct, Positive Control, Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy

    The full length PB1-F2 protein is necessary to inhibit NF-κB signaling. Vero cells were co-transfected with the NF-κB reporter, the different N-terminal (A) or C-terminal (B) PB1-F2 constructs or empty pcDNA4, the β-galactosidase control plasmid and 100 ng of pcDNA3-IKKβ. Cells transfected with the full length (FL) PB1-F2 Q/HK97 expression vector were used as a positive control for inhibition of NF-κB signalling. Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; *, P≤0.05; ****, P≤0.0001).
    Figure Legend Snippet: The full length PB1-F2 protein is necessary to inhibit NF-κB signaling. Vero cells were co-transfected with the NF-κB reporter, the different N-terminal (A) or C-terminal (B) PB1-F2 constructs or empty pcDNA4, the β-galactosidase control plasmid and 100 ng of pcDNA3-IKKβ. Cells transfected with the full length (FL) PB1-F2 Q/HK97 expression vector were used as a positive control for inhibition of NF-κB signalling. Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; *, P≤0.05; ****, P≤0.0001).

    Techniques Used: Transfection, Construct, Plasmid Preparation, Expressing, Positive Control, Inhibition, Luciferase

    PB1-F2 proteins from different influenza isolates inhibit NF-κB activation induced by IKKβ overexpression. Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid, the β-galactosidase control plasmid and 100 ng of pcDNA3-IKKβ (A) or 5 ng of pcDNA3-p65 (B). Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; ***, P≤0.001; ****, P≤0.0001).
    Figure Legend Snippet: PB1-F2 proteins from different influenza isolates inhibit NF-κB activation induced by IKKβ overexpression. Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid, the β-galactosidase control plasmid and 100 ng of pcDNA3-IKKβ (A) or 5 ng of pcDNA3-p65 (B). Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; ***, P≤0.001; ****, P≤0.0001).

    Techniques Used: Activation Assay, Over Expression, Transfection, Expressing, Plasmid Preparation, Luciferase

    PB1-F2 increases IKKβ kinase activity but inhibits NF-κB binding to DNA. A) Vero cells were transfected with empty pcDNA4 or pcDNA4-PB1-F2Q/HK97 and pcDNA3-IKKβ. Forty eight hours post transfection, cells were lysed and IKKβ was immunoprecipitated using rabbit anti-Flag antibody. The kinase assay was performed for the indicated times in the presence of 0.0925 MBq 32 P ATP, using GST-p65 and GST-IκBα as substrates. As a negative control (- C) non-transfected cells were used in the same experiment. Phosphorylation was detected by autoradiography. The levels of Flag-IKKβ present in the whole cell lysates (input) and immunoprecipitates (IP) were assessed by immunoblotting using rabbit anti-Flag antibody. B) Vero cells were transfected with pcDNA4-PB1-F2Q/HK97 and stimulated with 1 µg ml −1 LPS +800 ng ml −1 CD14 during one hour or left untreated (medium only). The cellular localisation of endogenous p65 was visualized using a rabbit anti-p65 antibody (green). Cells expressing PB1-F2 were identified (arrow) using a monoclonal mouse anti-myc antibody (red) and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). C) Vero cells were transfected with empty pcDNA4 or pcDNA4-PB1-F2Q/HK97 and pMACS K k .II plasmid. Cells expressing K k were selected using magnetic microbeads coated with anti-K k antibody. These cells were stimulated with 1 µg ml −1 LPS +800 ng ml −1 CD14 for one hour or left untreated (medium only). Nuclear extracts were prepared and incubated with a NF-κB biotinylated probe. Complexes were resolved in TBE polyacrylamide gel, transferred to a membrane and biotinylated DNA was detected by chemiluminescence.
    Figure Legend Snippet: PB1-F2 increases IKKβ kinase activity but inhibits NF-κB binding to DNA. A) Vero cells were transfected with empty pcDNA4 or pcDNA4-PB1-F2Q/HK97 and pcDNA3-IKKβ. Forty eight hours post transfection, cells were lysed and IKKβ was immunoprecipitated using rabbit anti-Flag antibody. The kinase assay was performed for the indicated times in the presence of 0.0925 MBq 32 P ATP, using GST-p65 and GST-IκBα as substrates. As a negative control (- C) non-transfected cells were used in the same experiment. Phosphorylation was detected by autoradiography. The levels of Flag-IKKβ present in the whole cell lysates (input) and immunoprecipitates (IP) were assessed by immunoblotting using rabbit anti-Flag antibody. B) Vero cells were transfected with pcDNA4-PB1-F2Q/HK97 and stimulated with 1 µg ml −1 LPS +800 ng ml −1 CD14 during one hour or left untreated (medium only). The cellular localisation of endogenous p65 was visualized using a rabbit anti-p65 antibody (green). Cells expressing PB1-F2 were identified (arrow) using a monoclonal mouse anti-myc antibody (red) and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). C) Vero cells were transfected with empty pcDNA4 or pcDNA4-PB1-F2Q/HK97 and pMACS K k .II plasmid. Cells expressing K k were selected using magnetic microbeads coated with anti-K k antibody. These cells were stimulated with 1 µg ml −1 LPS +800 ng ml −1 CD14 for one hour or left untreated (medium only). Nuclear extracts were prepared and incubated with a NF-κB biotinylated probe. Complexes were resolved in TBE polyacrylamide gel, transferred to a membrane and biotinylated DNA was detected by chemiluminescence.

    Techniques Used: Activity Assay, Binding Assay, Transfection, Immunoprecipitation, Kinase Assay, Negative Control, Autoradiography, Expressing, Staining, Plasmid Preparation, Incubation

    PB1-F2 modulates NF-κB signaling. A) Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid along with the β-galactosidase control plasmid. Forty eight hours post-transfection, the cells were either induced or not induced with 25 µg ml −1 Poly (I:C), 20 ng ml −1 TNFα, 1 µg ml −1 LPS or 10 ng ml −1 IL-1β for five hours, cell extracts were made and the luciferase and galactosidase activites were measured. B) Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid, the β-galactosidase control plasmid and 100 ng of TRIF, Myd88 or MAVS expressing vectors. Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001). C) Vero cells in a 6 well plate, were transfected with 3 µg of each of the different PB1-F2 constructs or the empty pcDNA4 plasmid. Forty eight hours post-transfection, the cells were lysed and PB1-F2 expression was detected by immunoblotting using a monoclonal mouse anti-myc antibody.
    Figure Legend Snippet: PB1-F2 modulates NF-κB signaling. A) Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid along with the β-galactosidase control plasmid. Forty eight hours post-transfection, the cells were either induced or not induced with 25 µg ml −1 Poly (I:C), 20 ng ml −1 TNFα, 1 µg ml −1 LPS or 10 ng ml −1 IL-1β for five hours, cell extracts were made and the luciferase and galactosidase activites were measured. B) Vero cells were co-transfected with the NF-κB reporter and either each of the different PB1-F2 expression vectors or the empty pcDNA4 plasmid, the β-galactosidase control plasmid and 100 ng of TRIF, Myd88 or MAVS expressing vectors. Forty eight hours post-transfection, the cells were lysed and the luciferase and galactosidase activities were measured. Data are representative of at least 3 independent experiments. Bars represent average values and standard deviations of firefly luciferase activities from triplicate samples normalized to the expression of galactosidase. Asterisks indicate results significantly different from control empty vector (one-way ANOVA followed by Dunnett's test; *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001). C) Vero cells in a 6 well plate, were transfected with 3 µg of each of the different PB1-F2 constructs or the empty pcDNA4 plasmid. Forty eight hours post-transfection, the cells were lysed and PB1-F2 expression was detected by immunoblotting using a monoclonal mouse anti-myc antibody.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Construct

    10) Product Images from "Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton"

    Article Title: Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton

    Journal: Antiviral Research

    doi: 10.1016/j.antiviral.2005.02.003

    Anti-viral activity of Sukumo extract. (A) Anti-HIV-1 activity of Sukumo extract in MT-4 cells was measured by MTT assay. HIV-1 (III B ) was used in this study ((●) mock infected and (♦) HIV-1)) and the EC 50 values for inhibition of Sukumo extract against HIV-1 replication were determined. (B) Anti-HSV-1 activity of Sukumo extract in Vero cells was determined by plaque assay. The results shown are mean ± S.D. of triplicates.
    Figure Legend Snippet: Anti-viral activity of Sukumo extract. (A) Anti-HIV-1 activity of Sukumo extract in MT-4 cells was measured by MTT assay. HIV-1 (III B ) was used in this study ((●) mock infected and (♦) HIV-1)) and the EC 50 values for inhibition of Sukumo extract against HIV-1 replication were determined. (B) Anti-HSV-1 activity of Sukumo extract in Vero cells was determined by plaque assay. The results shown are mean ± S.D. of triplicates.

    Techniques Used: Activity Assay, MTT Assay, Infection, Inhibition, Plaque Assay

    11) Product Images from "Expression of an Epitope-Tagged Virulence Protein in Rickettsia parkeri Using Transposon Insertion"

    Article Title: Expression of an Epitope-Tagged Virulence Protein in Rickettsia parkeri Using Transposon Insertion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037310

    Detection of FLAG-RickA in bacteria. R. parkeri strains that were not transformed ( Rp ) or transformed with pMW1650-FLAG-RickA ( Rp FLAG-RickA) were used to infect Vero cells and then (A) labeled by immunofluorescence with anti-RickA antibody and stained for DNA with DAPI, or (B) labeled by immunofluorescence with anti-FLAG antibody and stained for DNA with DAPI. In the merged images, RickA or FLAG are labeled in green, and DNA in red. Scale bar 10 µm. Higher magnification images of individual bacteria (highlighted in boxes in the lower magnification images) are shown on the right.
    Figure Legend Snippet: Detection of FLAG-RickA in bacteria. R. parkeri strains that were not transformed ( Rp ) or transformed with pMW1650-FLAG-RickA ( Rp FLAG-RickA) were used to infect Vero cells and then (A) labeled by immunofluorescence with anti-RickA antibody and stained for DNA with DAPI, or (B) labeled by immunofluorescence with anti-FLAG antibody and stained for DNA with DAPI. In the merged images, RickA or FLAG are labeled in green, and DNA in red. Scale bar 10 µm. Higher magnification images of individual bacteria (highlighted in boxes in the lower magnification images) are shown on the right.

    Techniques Used: Transformation Assay, Labeling, Immunofluorescence, Staining

    12) Product Images from "Characterization of Glycoprotein-Mediated Entry of Severe Fever with Thrombocytopenia Syndrome Virus"

    Article Title: Characterization of Glycoprotein-Mediated Entry of Severe Fever with Thrombocytopenia Syndrome Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.00110-16

    Inhibition of SFTSVpv and SFTSV infection by H + -ATPase inhibitors. Huh7 or Vero cells were inoculated with SFTSVpv, RVFVpv, VSVpv, or MLVpv (A) or with SFTSV (B) after treatment of these cells with various concentrations of ammonium chloride (left) or
    Figure Legend Snippet: Inhibition of SFTSVpv and SFTSV infection by H + -ATPase inhibitors. Huh7 or Vero cells were inoculated with SFTSVpv, RVFVpv, VSVpv, or MLVpv (A) or with SFTSV (B) after treatment of these cells with various concentrations of ammonium chloride (left) or

    Techniques Used: Inhibition, Infection

    Expression and localization of SFTSV-GP. (A) Expression of SFTSV-GP in Vero (top) or Huh7 (bottom) cells infected with SFTSV (left) or transfected with a GP plasmid (right) was examined by an immunofluorescence assay with an anti-SFTSV-GP monoclonal antibody.
    Figure Legend Snippet: Expression and localization of SFTSV-GP. (A) Expression of SFTSV-GP in Vero (top) or Huh7 (bottom) cells infected with SFTSV (left) or transfected with a GP plasmid (right) was examined by an immunofluorescence assay with an anti-SFTSV-GP monoclonal antibody.

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

    Effects of 25HC on infection by SFTSVpv and SFTSV. (A) Inhibition of SFTSVpv infection by 25HC. Vero (left) and Huh7 (right) cells were inoculated with pseudotype viruses after treatment of these cells with various concentrations of 25HC. Infectivities
    Figure Legend Snippet: Effects of 25HC on infection by SFTSVpv and SFTSV. (A) Inhibition of SFTSVpv infection by 25HC. Vero (left) and Huh7 (right) cells were inoculated with pseudotype viruses after treatment of these cells with various concentrations of 25HC. Infectivities

    Techniques Used: Infection, Inhibition

    13) Product Images from "Caspase-Mediated Cleavage of Nucleocapsid protein of a Protease-Independent Porcine Epidemic Diarrhea Virus Strain"

    Article Title: Caspase-Mediated Cleavage of Nucleocapsid protein of a Protease-Independent Porcine Epidemic Diarrhea Virus Strain

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.198026

    Subcellular localization of PEDV N protein in Vero cells. Vero cells were mock-infected or infected at MOI of 1 with PEDV 8aa with DMSO, 8aa with Z-VAD-fmk (100 μM) or KD with trypsin (1 µg/ml). For the expression of N protein, 24 h-old semi-confluent cells were transfected with pIRES-N-nHA, and cells were incubated at 37 °C for (A) 12 h (KD) or 36 h (8aa and N transfection), or (B)12, 24 or 48 h (8aa and 8aa with Z-VAD-fmk). Fixed and permeabilized cells were stained with anti-PEDV N monoclonal antibody (green) and SYTOX orange (Blue) and examined by confocal microscopy.
    Figure Legend Snippet: Subcellular localization of PEDV N protein in Vero cells. Vero cells were mock-infected or infected at MOI of 1 with PEDV 8aa with DMSO, 8aa with Z-VAD-fmk (100 μM) or KD with trypsin (1 µg/ml). For the expression of N protein, 24 h-old semi-confluent cells were transfected with pIRES-N-nHA, and cells were incubated at 37 °C for (A) 12 h (KD) or 36 h (8aa and N transfection), or (B)12, 24 or 48 h (8aa and 8aa with Z-VAD-fmk). Fixed and permeabilized cells were stained with anti-PEDV N monoclonal antibody (green) and SYTOX orange (Blue) and examined by confocal microscopy.

    Techniques Used: Infection, Expressing, Transfection, Incubation, Staining, Confocal Microscopy

    14) Product Images from "A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes"

    Article Title: A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00693

    Extracellular amastigotes release Ssp-4 associated with vesicles interacting with host cell. (A) Scanning electron microscopy of extracellular amastigotes (EAs) adhered to coverslips coated with poly-L-lysine or Vero cells. Scale bars: 2 and 5 μm. (B) Transmission electron microscopy of HeLa cells incubated for 30 min with EAs from the G strain (colored in red). Scale bar: 1 μm. Black arrows indicate EA secreted vesicles. (C) Immunofluorescence of HeLa cells incubated for 30 min with EAs from G (MOI 10:1) and mAb1D9 (green), DAPI (blue), and phalloidin-TRITC (red). Upper panels: images of a focal plane showing vesicles secreted by EAs of the G strain (green) associated with actin (red); bottom panels: three-dimensional reconstruction from a Z -series acquired by a confocal microscope. Scale bar: 2 μm. Arrows indicate vesicular structures secreted by EAs. (D) Extracellular amastigotes (EAs) from the G and Y strains were incubated for 6 h in RPMI medium without fetal bovine serum. Supernatant was collected and fractionated in three different populations: V2 (pelleted after 2 h of ultracentrifugation), V16 (pelleted after 16 h ultracentrifugation), and VF (supernatant from pellet V16). Western blotting with mAb1D9 and silver-stained SDS–PAGE of the different fractions. Whole cell (WC) lysate of EAs of the G strain was used as control.
    Figure Legend Snippet: Extracellular amastigotes release Ssp-4 associated with vesicles interacting with host cell. (A) Scanning electron microscopy of extracellular amastigotes (EAs) adhered to coverslips coated with poly-L-lysine or Vero cells. Scale bars: 2 and 5 μm. (B) Transmission electron microscopy of HeLa cells incubated for 30 min with EAs from the G strain (colored in red). Scale bar: 1 μm. Black arrows indicate EA secreted vesicles. (C) Immunofluorescence of HeLa cells incubated for 30 min with EAs from G (MOI 10:1) and mAb1D9 (green), DAPI (blue), and phalloidin-TRITC (red). Upper panels: images of a focal plane showing vesicles secreted by EAs of the G strain (green) associated with actin (red); bottom panels: three-dimensional reconstruction from a Z -series acquired by a confocal microscope. Scale bar: 2 μm. Arrows indicate vesicular structures secreted by EAs. (D) Extracellular amastigotes (EAs) from the G and Y strains were incubated for 6 h in RPMI medium without fetal bovine serum. Supernatant was collected and fractionated in three different populations: V2 (pelleted after 2 h of ultracentrifugation), V16 (pelleted after 16 h ultracentrifugation), and VF (supernatant from pellet V16). Western blotting with mAb1D9 and silver-stained SDS–PAGE of the different fractions. Whole cell (WC) lysate of EAs of the G strain was used as control.

    Techniques Used: Electron Microscopy, Transmission Assay, Incubation, Immunofluorescence, Microscopy, Western Blot, Staining, SDS Page

    15) Product Images from "A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway"

    Article Title: A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

    Journal: Journal of Virology

    doi: 10.1128/JVI.01237-15

    IBV E is present in two pools in infected cells and virions, with the majority being in the HMW pool. (A) Vero cells infected with IBV were lysed at 8 h postinfection and run on a 5 to 20% sucrose gradient, or virions were purified from infected cell
    Figure Legend Snippet: IBV E is present in two pools in infected cells and virions, with the majority being in the HMW pool. (A) Vero cells infected with IBV were lysed at 8 h postinfection and run on a 5 to 20% sucrose gradient, or virions were purified from infected cell

    Techniques Used: Infection, Purification

    16) Product Images from "Temporal Transcriptome and Promoter Architecture of the African Swine Fever Virus"

    Article Title: Temporal Transcriptome and Promoter Architecture of the African Swine Fever Virus

    Journal: bioRxiv

    doi: 10.1101/847343

    Early and late gene expression of ASFV BA71V. (a) FPKM values for 20 most highly expressed ASFV TUs according to CAGE-seq at 5h (left) and 16h (right) post-infection, RNA-seq equivalent results found in SUPPLEMENTARY Figure 3. Genes highlighted in maroon indicate those encoding proteins which were also found in the 20 most-abundantly expressed AFSV proteins during infection of either WSL-HP, HEK293 or Vero cells according to proteome analysis done by Keßler et al . 31 . Gene functions are shown after their name with TR and PSP referring to predicted transmembrane region and putative signal peptide, respectively. (b) MAplot from DESeq2 analysis of CAGE-seq representing the DESeq2 base mean of transcript levels versus their log2 fold change, with significantly differentially expressed genes in purple (adjusted p -value
    Figure Legend Snippet: Early and late gene expression of ASFV BA71V. (a) FPKM values for 20 most highly expressed ASFV TUs according to CAGE-seq at 5h (left) and 16h (right) post-infection, RNA-seq equivalent results found in SUPPLEMENTARY Figure 3. Genes highlighted in maroon indicate those encoding proteins which were also found in the 20 most-abundantly expressed AFSV proteins during infection of either WSL-HP, HEK293 or Vero cells according to proteome analysis done by Keßler et al . 31 . Gene functions are shown after their name with TR and PSP referring to predicted transmembrane region and putative signal peptide, respectively. (b) MAplot from DESeq2 analysis of CAGE-seq representing the DESeq2 base mean of transcript levels versus their log2 fold change, with significantly differentially expressed genes in purple (adjusted p -value

    Techniques Used: Expressing, Infection, RNA Sequencing Assay

    17) Product Images from "Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling"

    Article Title: Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148560

    The reduction of total and phosphorylated STAT1 in NDV-infected cells was inhibited after treatment with Ub E1 inhibitor PYR-41 at different time points. (A) STAT1 and phospho-STAT1 expression levels in PYR-41 treated Vero cells at 6 hpi. (B) Phospho-STAT1 levels in PYR-41 treated Vero cells at 4, 6, 8 hpi. (C) Exogenous mutant STAT1 lacking 701aa phosphorylation site were not degraded in the course of NDV infection. One microgram pFlag-STAT1 or pFlag-Y701F was transfected into A549 cells cultured in 6-well plates. At 12 h post transfection, the cells were subsequently infected with NDVs at a MOI of 3. The cells were harvested at 24 hpi. (D) The expression levels of IFN-responsive genes in V-expressing A549 cells after stimulation of IFN-α. A549 cells in 6-well plates were transfected with 3 μg pCI-V or pCI-neo for each well as above-described. At 4 h and 8 h post-transfection, the cells were harvested following the treatment with 500 U/ml IFN-α for 30 min.
    Figure Legend Snippet: The reduction of total and phosphorylated STAT1 in NDV-infected cells was inhibited after treatment with Ub E1 inhibitor PYR-41 at different time points. (A) STAT1 and phospho-STAT1 expression levels in PYR-41 treated Vero cells at 6 hpi. (B) Phospho-STAT1 levels in PYR-41 treated Vero cells at 4, 6, 8 hpi. (C) Exogenous mutant STAT1 lacking 701aa phosphorylation site were not degraded in the course of NDV infection. One microgram pFlag-STAT1 or pFlag-Y701F was transfected into A549 cells cultured in 6-well plates. At 12 h post transfection, the cells were subsequently infected with NDVs at a MOI of 3. The cells were harvested at 24 hpi. (D) The expression levels of IFN-responsive genes in V-expressing A549 cells after stimulation of IFN-α. A549 cells in 6-well plates were transfected with 3 μg pCI-V or pCI-neo for each well as above-described. At 4 h and 8 h post-transfection, the cells were harvested following the treatment with 500 U/ml IFN-α for 30 min.

    Techniques Used: Infection, Expressing, Mutagenesis, Transfection, Cell Culture

    STAT1 expression in NDV infected or V-expressing plasmids transfected cells. (A) No STAT1 reduction was observed in Vero cells infected with NDV ZJ1, 9a5b or LaSota at MOI 3 at 6, 12 or 24 hpi. (B) Over-expression of ZJ1, 9a5b and LaSota V protein did not effect on STAT1 expression in Vero cells transfected with V-expressing plasmids. (C) STAT1 was reduced at 48 h post-transfection in NDV-infected Vero cells (MOI = 3) transfected in advance with V-expressing plasmids. (D) STAT1 expression in A549 cells transfected with V expressing plasmids or infected with ZJ1 or LaSota was detected at 48 h post-transfection or 12 h post-infection by indirect fluorescence assay. These infected A549 cells were fixed and detected for P/V/W proteins with a mixture of anti-serum Pab-V1 and Pab-V2; while the presence of STAT1 was determined by anti-STAT1 antibody (ab31369). Cellular nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).
    Figure Legend Snippet: STAT1 expression in NDV infected or V-expressing plasmids transfected cells. (A) No STAT1 reduction was observed in Vero cells infected with NDV ZJ1, 9a5b or LaSota at MOI 3 at 6, 12 or 24 hpi. (B) Over-expression of ZJ1, 9a5b and LaSota V protein did not effect on STAT1 expression in Vero cells transfected with V-expressing plasmids. (C) STAT1 was reduced at 48 h post-transfection in NDV-infected Vero cells (MOI = 3) transfected in advance with V-expressing plasmids. (D) STAT1 expression in A549 cells transfected with V expressing plasmids or infected with ZJ1 or LaSota was detected at 48 h post-transfection or 12 h post-infection by indirect fluorescence assay. These infected A549 cells were fixed and detected for P/V/W proteins with a mixture of anti-serum Pab-V1 and Pab-V2; while the presence of STAT1 was determined by anti-STAT1 antibody (ab31369). Cellular nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).

    Techniques Used: Expressing, Infection, Transfection, Over Expression, Fluorescence, Staining

    Newcastle disease virus infection impaired IFN-α-induced STAT1 phosphorylation. (A) IFN-α-induced phosphorylated STAT1 degradation in NDV-infected A549 and Vero cells. A549 or Vero cells were infected with NDV strains ZJ1 at MOI 3. At indicated time points post infection, A549 and Vero cells were stimulated with 500 U/ml human IFN-α or IFN-γ in 1 ml DMEM at 37°C for 15 min. Uninfected cells were stimulated with IFN as negative controls (IFNα+/infection-). IFN-α-induced phospho-STAT1 was observed (IFNα+/infection+) for reduction in total STAT1 proteins. IFN-γ-induced phospho-STAT1 proteins were not reduced. (B) Phospho-STAT1 in A549 cells transfected with V-expressing plasmids after stimulation with IFN-α. (C) Expression level of STAT1 and phospho-STAT1 decreased in V-expressing Vero cells after IFN-α stimulation. Vero cells were mock transfected with pCI-neo plasmids. Cells on glass coverlips were transfected with pCI-V/ZJ1 plasmids. At 48 h post-transfection, cells were treated with IFN-α for 15 min prior to fixation as in “Material and methods”. V protein was detected by a mixture of anti-serum Pab-V1 and Pab-V2; STAT1 and phosphorylation were determined by anti-STAT1 antibody (ab31369) and anti-phospho-STAT1 antibody (ab30645). Cellular nuclei were stained with DAPI.
    Figure Legend Snippet: Newcastle disease virus infection impaired IFN-α-induced STAT1 phosphorylation. (A) IFN-α-induced phosphorylated STAT1 degradation in NDV-infected A549 and Vero cells. A549 or Vero cells were infected with NDV strains ZJ1 at MOI 3. At indicated time points post infection, A549 and Vero cells were stimulated with 500 U/ml human IFN-α or IFN-γ in 1 ml DMEM at 37°C for 15 min. Uninfected cells were stimulated with IFN as negative controls (IFNα+/infection-). IFN-α-induced phospho-STAT1 was observed (IFNα+/infection+) for reduction in total STAT1 proteins. IFN-γ-induced phospho-STAT1 proteins were not reduced. (B) Phospho-STAT1 in A549 cells transfected with V-expressing plasmids after stimulation with IFN-α. (C) Expression level of STAT1 and phospho-STAT1 decreased in V-expressing Vero cells after IFN-α stimulation. Vero cells were mock transfected with pCI-neo plasmids. Cells on glass coverlips were transfected with pCI-V/ZJ1 plasmids. At 48 h post-transfection, cells were treated with IFN-α for 15 min prior to fixation as in “Material and methods”. V protein was detected by a mixture of anti-serum Pab-V1 and Pab-V2; STAT1 and phosphorylation were determined by anti-STAT1 antibody (ab31369) and anti-phospho-STAT1 antibody (ab30645). Cellular nuclei were stained with DAPI.

    Techniques Used: Infection, Transfection, Expressing, Staining

    18) Product Images from "Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes"

    Article Title: Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

    Journal: Cytotechnology

    doi: 10.1007/s10616-011-9421-2

    Microscopic observation of Vero cells attached on microcarriers. Evolution of Vero cell morphology on microporous microcarriers at 4 h ( A ), 56 h ( B ), 70 h ( C ) and 94 h ( D ) after cell seeding, during culture performed with
    Figure Legend Snippet: Microscopic observation of Vero cells attached on microcarriers. Evolution of Vero cell morphology on microporous microcarriers at 4 h ( A ), 56 h ( B ), 70 h ( C ) and 94 h ( D ) after cell seeding, during culture performed with

    Techniques Used:

    Evolution of the permittivity and the specific permittivity, ε X , with the adhered Vero cell concentration. A Correlation between permittivity and concentration of Vero cells attached on microcarriers, during Batch 1 (B1: black circle ), Batch
    Figure Legend Snippet: Evolution of the permittivity and the specific permittivity, ε X , with the adhered Vero cell concentration. A Correlation between permittivity and concentration of Vero cells attached on microcarriers, during Batch 1 (B1: black circle ), Batch

    Techniques Used: Concentration Assay

    19) Product Images from "The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection"

    Article Title: The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.81

    ASFV induces the expression of ER chaperones but not the expression of ERp57 or fragmentation of Bap31. ( a ) Western blot analysis of the ER chaperones in Vero cells infected with ASFV or treated with tunicamycin (Tm). Cell lysates were harvested at the indicated times after infection and then, analyzed. Actin was used as protein load control and viral infection was followed by p30 viral protein expression. ( b ) Quantification of the bands by densitometry corrected to actin data and normalized to control values. ( c ) Representative confocal micrographs of Vero cells grown on glass slides were treated with tunicamycin or infected with B54GFP-2 for 16 hpi. Cells were fixed, stained with anti-BiP plus Alexa Fluor 594-conjugated secondary antibody and incubated with Topro-3 for DNA staining and then analyzed by confocal fluorescence microscopy. Bar 10 μ M
    Figure Legend Snippet: ASFV induces the expression of ER chaperones but not the expression of ERp57 or fragmentation of Bap31. ( a ) Western blot analysis of the ER chaperones in Vero cells infected with ASFV or treated with tunicamycin (Tm). Cell lysates were harvested at the indicated times after infection and then, analyzed. Actin was used as protein load control and viral infection was followed by p30 viral protein expression. ( b ) Quantification of the bands by densitometry corrected to actin data and normalized to control values. ( c ) Representative confocal micrographs of Vero cells grown on glass slides were treated with tunicamycin or infected with B54GFP-2 for 16 hpi. Cells were fixed, stained with anti-BiP plus Alexa Fluor 594-conjugated secondary antibody and incubated with Topro-3 for DNA staining and then analyzed by confocal fluorescence microscopy. Bar 10 μ M

    Techniques Used: Expressing, Western Blot, Infection, Staining, Incubation, Fluorescence, Microscopy

    ASFV regulates UPR signaling. ( a ) Western blot analysis of CHOP, GADD34, ATF4, p30 and p72 viral proteins in Vero cells infected with ASFV or treated with tunicamycin, lysated at the indicated times after infection with actin as protein load control. ( b ) Quantification of the bands corresponding to GADD34 by densitometry was corrected to actin data and normalized to control values ( c ) Unspliced (uXBP1) and spliced (sXBP1) bands were RT-PCR-amplified using specific primer pairs. Cells treated with tunicamycin were used as a positive control for the induction of spliced XBP1. ( d ) XBP1 mRNA levels quantified with real-time RT-PCR. Total RNA was isolated from ASFV-infected Vero cells at a range of times post-infection. The XBP1 message was normalized to the 18S ribosomal message and x-fold changes were calculated as described in Materials and Methods. DTT and Tm-treated cells were used as control. Data are means±S.D. from three independent experiments
    Figure Legend Snippet: ASFV regulates UPR signaling. ( a ) Western blot analysis of CHOP, GADD34, ATF4, p30 and p72 viral proteins in Vero cells infected with ASFV or treated with tunicamycin, lysated at the indicated times after infection with actin as protein load control. ( b ) Quantification of the bands corresponding to GADD34 by densitometry was corrected to actin data and normalized to control values ( c ) Unspliced (uXBP1) and spliced (sXBP1) bands were RT-PCR-amplified using specific primer pairs. Cells treated with tunicamycin were used as a positive control for the induction of spliced XBP1. ( d ) XBP1 mRNA levels quantified with real-time RT-PCR. Total RNA was isolated from ASFV-infected Vero cells at a range of times post-infection. The XBP1 message was normalized to the 18S ribosomal message and x-fold changes were calculated as described in Materials and Methods. DTT and Tm-treated cells were used as control. Data are means±S.D. from three independent experiments

    Techniques Used: Western Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Quantitative RT-PCR, Isolation

    Caspase 3 activation and membrane blebbing enhance ASFV spread. Vero cells treated with DEVD-CHO, Y-27632 or Blebbistatin were infected with ASFV. Drugs were added 2 h before infection or 6 h after, as indicated. At 48 hpi, cells and media were harvested and titrated by plaque assay. Total ( a ) or extracellular virus titers ( b ) are shown. Error bars indicate S.D. from three independent experiments. Statistically significant differences are indicated by asterisks (* P
    Figure Legend Snippet: Caspase 3 activation and membrane blebbing enhance ASFV spread. Vero cells treated with DEVD-CHO, Y-27632 or Blebbistatin were infected with ASFV. Drugs were added 2 h before infection or 6 h after, as indicated. At 48 hpi, cells and media were harvested and titrated by plaque assay. Total ( a ) or extracellular virus titers ( b ) are shown. Error bars indicate S.D. from three independent experiments. Statistically significant differences are indicated by asterisks (* P

    Techniques Used: Activation Assay, Infection, Plaque Assay

    ASFV infection is dependent on ATF6 activation. ( a ) ASFV-infected cell percentages in Vero cells pretreated with AEBSF at the indicated concentrations and then infected. Infected cells were detected by FACS and data normalized to infection rates in untreated cells. Representative FACS profiles obtained during the analysis are shown. Infected cells were gated in M1 and expressed as a percentage of total cell analyzed. ( b ) Caspase activities at 24 hpi in Vero cells pretreated with 300 μ M AEBSF and then infected. Data were normalized to mock-infected control values. Cells treated with Staurosporine (Stau) were used as control. Asterisks denote statistically significant differences (** P
    Figure Legend Snippet: ASFV infection is dependent on ATF6 activation. ( a ) ASFV-infected cell percentages in Vero cells pretreated with AEBSF at the indicated concentrations and then infected. Infected cells were detected by FACS and data normalized to infection rates in untreated cells. Representative FACS profiles obtained during the analysis are shown. Infected cells were gated in M1 and expressed as a percentage of total cell analyzed. ( b ) Caspase activities at 24 hpi in Vero cells pretreated with 300 μ M AEBSF and then infected. Data were normalized to mock-infected control values. Cells treated with Staurosporine (Stau) were used as control. Asterisks denote statistically significant differences (** P

    Techniques Used: Infection, Activation Assay, FACS

    ASFV infection is independent of caspase 12 activation. Vero cells were pretreated with Z-ATAD-FMK and then infected with ASFV. ( a ) Infected cells were detected by FACS and percentages normalized to values in untreated cells. Representative FACS profiles are shown in the graphs below. Infected cells were gated in M1 and expressed as a percentage of total cells analyzed. ( b ) Cells and media were harvested at 48 hpi and titrated by plaque assay. Intracellular (I) or extracellular (E) virus titers in pfu/ml are shown. ( c ) Caspase activities were analyzed at 24 hpi using specific activity assays. Data were normalized to control values. Error bars indicate S.D. from three independent experiments and statistically significant differences are indicated by asterisks (** P
    Figure Legend Snippet: ASFV infection is independent of caspase 12 activation. Vero cells were pretreated with Z-ATAD-FMK and then infected with ASFV. ( a ) Infected cells were detected by FACS and percentages normalized to values in untreated cells. Representative FACS profiles are shown in the graphs below. Infected cells were gated in M1 and expressed as a percentage of total cells analyzed. ( b ) Cells and media were harvested at 48 hpi and titrated by plaque assay. Intracellular (I) or extracellular (E) virus titers in pfu/ml are shown. ( c ) Caspase activities were analyzed at 24 hpi using specific activity assays. Data were normalized to control values. Error bars indicate S.D. from three independent experiments and statistically significant differences are indicated by asterisks (** P

    Techniques Used: Infection, Activation Assay, FACS, Plaque Assay, Activity Assay

    Caspase activities in ASFV-infected cells. Caspase activities were measured in virus-infected cell lysates using specific activity assays at the indicated time points after infection. Data were normalized to the corresponding values for mock-infected cells for each caspase. ( a and b ) Caspase 3 and 9 activity of virus-infected Vero cells. ( c and d ) Caspase 8 activity of ASFV-infected Vero and WSL-R macrophage cell lines, respectively. ( e ) Caspase 12 activity of ASFV-infected Vero cells. ( f ) Representative confocal micrographs of nuclear localization of caspase 12 in ASFV-infected Vero cells. Cells treated with tunicamycin were used as controls. Statistical significance is indicated by asterisks (* P
    Figure Legend Snippet: Caspase activities in ASFV-infected cells. Caspase activities were measured in virus-infected cell lysates using specific activity assays at the indicated time points after infection. Data were normalized to the corresponding values for mock-infected cells for each caspase. ( a and b ) Caspase 3 and 9 activity of virus-infected Vero cells. ( c and d ) Caspase 8 activity of ASFV-infected Vero and WSL-R macrophage cell lines, respectively. ( e ) Caspase 12 activity of ASFV-infected Vero cells. ( f ) Representative confocal micrographs of nuclear localization of caspase 12 in ASFV-infected Vero cells. Cells treated with tunicamycin were used as controls. Statistical significance is indicated by asterisks (* P

    Techniques Used: Infection, Activity Assay

    ASFV infection activates the ATF6 pathway. ( a ) Representative confocal micrographs of Vero cells transfected with the GFP-ATF6 plasmid. After 24 h, untreated cells showed a typical ER pattern, while in cells treated with tunicamicyn, GFP-ATF6 relocated to the Golgi ( b ) and to the nucleus ( c ). ( d–f ) Representative confocal micrographs of ASFV-infected transfected cells. ATF6 translocated to the viral factories and to the nucleus. ( d , arrow) A neighboring uninfected cell is shown for comparison. Bar 25 μ m. ( g ) Percentages of GFP-ATF6 subcellular localization events in infected and tunicamycin treated cells in triplicate experiments calculated in a total of 5 × 10 4 cells/well
    Figure Legend Snippet: ASFV infection activates the ATF6 pathway. ( a ) Representative confocal micrographs of Vero cells transfected with the GFP-ATF6 plasmid. After 24 h, untreated cells showed a typical ER pattern, while in cells treated with tunicamicyn, GFP-ATF6 relocated to the Golgi ( b ) and to the nucleus ( c ). ( d–f ) Representative confocal micrographs of ASFV-infected transfected cells. ATF6 translocated to the viral factories and to the nucleus. ( d , arrow) A neighboring uninfected cell is shown for comparison. Bar 25 μ m. ( g ) Percentages of GFP-ATF6 subcellular localization events in infected and tunicamycin treated cells in triplicate experiments calculated in a total of 5 × 10 4 cells/well

    Techniques Used: Infection, Transfection, Plasmid Preparation

    20) Product Images from "Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase"

    Article Title: Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase

    Journal: Virus Research

    doi: 10.1016/j.virusres.2018.05.019

    G0/G1, G1/S and G2/M phases synchronization treatment. (A) Serum-starved Vero cells were mock infected or infected with PEDV. Cell cycle profiles at 18 h p.i. were determined by FACS analysis. (B) Cell cycle profiles of Vero cells synchronized at the G1/S phase border using double-thymidine treatment. Following synchronization (time 0) cells were released from block and simultaneously mock infected and infected with PEDV, and cell cycle profiles were analyzed at 18 h p.i.. (C) Asynchronously growing Vero cells were treated with nocodazole for 16 h. Following synchronization (time 0) cells were released from block and simultaneously mock-infected and infected with PEDV. Cell cycle profiles were analyzed at 18 h p.i.. The results are shown as mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: G0/G1, G1/S and G2/M phases synchronization treatment. (A) Serum-starved Vero cells were mock infected or infected with PEDV. Cell cycle profiles at 18 h p.i. were determined by FACS analysis. (B) Cell cycle profiles of Vero cells synchronized at the G1/S phase border using double-thymidine treatment. Following synchronization (time 0) cells were released from block and simultaneously mock infected and infected with PEDV, and cell cycle profiles were analyzed at 18 h p.i.. (C) Asynchronously growing Vero cells were treated with nocodazole for 16 h. Following synchronization (time 0) cells were released from block and simultaneously mock-infected and infected with PEDV. Cell cycle profiles were analyzed at 18 h p.i.. The results are shown as mean ± SEM of three independent experiments. * P

    Techniques Used: Infection, FACS, Blocking Assay

    21) Product Images from "Chemical Induction of Endogenous Retrovirus Particles from the Vero Cell Line of African Green Monkeys ▿"

    Article Title: Chemical Induction of Endogenous Retrovirus Particles from the Vero Cell Line of African Green Monkeys ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00147-11

    Drug dose evaluation and PERT activity. Drug dose range for IUdR (A), AzaC (B), and NaBut (C) was determined by evaluating the Vero cell viability after drug treatment with various concentrations for 48 h. Cell toxicity was determined at day 0 (day of
    Figure Legend Snippet: Drug dose evaluation and PERT activity. Drug dose range for IUdR (A), AzaC (B), and NaBut (C) was determined by evaluating the Vero cell viability after drug treatment with various concentrations for 48 h. Cell toxicity was determined at day 0 (day of

    Techniques Used: Activity Assay

    Infectivity analysis of drug-induced retrovirus from Vero cells. Vero cells (1 × 10 6 ) were planted in 25-cm 2 flasks for 16 h and treated with 1.25 μg/ml AzaC or 200 μg/ml IUdR for 48 h. In the case of one flask, medium was replaced
    Figure Legend Snippet: Infectivity analysis of drug-induced retrovirus from Vero cells. Vero cells (1 × 10 6 ) were planted in 25-cm 2 flasks for 16 h and treated with 1.25 μg/ml AzaC or 200 μg/ml IUdR for 48 h. In the case of one flask, medium was replaced

    Techniques Used: Infection

    22) Product Images from "Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion"

    Article Title: Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

    Journal: Journal of Virology

    doi: 10.1128/JVI.00886-12

    NDV resists complement-mediated neutralization in a species-specific manner. Purified NDV/HeLa, NDV/Vero, NDV/CHO K1, NDV/CHO 5.3, and NDV/egg were incubated for 1 h at 37°C with 1:10, 1:20, and 1:40 dilutions of normal human serum (NHS) (A) and
    Figure Legend Snippet: NDV resists complement-mediated neutralization in a species-specific manner. Purified NDV/HeLa, NDV/Vero, NDV/CHO K1, NDV/CHO 5.3, and NDV/egg were incubated for 1 h at 37°C with 1:10, 1:20, and 1:40 dilutions of normal human serum (NHS) (A) and

    Techniques Used: Neutralization, Purification, Incubation

    Incorporation of RCA molecules by NDV grown in mammalian cells. (A) Lysates of CHO K1, CHO 5.3, HeLa, and Vero cells and the purified viruses grown in them were immunoblotted with antibodies to CD46 and CD55. Protein bands of actin and the NDV matrix
    Figure Legend Snippet: Incorporation of RCA molecules by NDV grown in mammalian cells. (A) Lysates of CHO K1, CHO 5.3, HeLa, and Vero cells and the purified viruses grown in them were immunoblotted with antibodies to CD46 and CD55. Protein bands of actin and the NDV matrix

    Techniques Used: Purification

    NDV grown in mammalian cells acquire human C3b cofactor activity. Purified NDV/HeLa (A), NDV/Vero (B), and NDV/egg (C) were incubated for 1 to 24 h with purified human complement component C3b and factor I. Lane 1, molecular mass marker; lanes 2 to 5,
    Figure Legend Snippet: NDV grown in mammalian cells acquire human C3b cofactor activity. Purified NDV/HeLa (A), NDV/Vero (B), and NDV/egg (C) were incubated for 1 to 24 h with purified human complement component C3b and factor I. Lane 1, molecular mass marker; lanes 2 to 5,

    Techniques Used: Activity Assay, Purification, Incubation, Marker

    23) Product Images from "A plasmid-based reporter system for live cell imaging of dengue infected cells"

    Article Title: A plasmid-based reporter system for live cell imaging of dengue infected cells

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2014.10.010

    Kinetics of nuclear localization of GFP after DENV infection. Vero cells transfected with p4B5-EGFP were infected with DENV-2 16681 and sequential images were acquired to assess the time for GFP to localize into the nucleus. Each set of images shows the expression of GFP in p4B5-EGFP transfected cells (top row), nuclear stain using NucBlue (middle row) and transmitted images (bottom row) at each time point. Time after addition of virus to the culture is located at the lower left of each image (magnification, ×20). Nuclear/total fluorescence (N/T) was calculated at each time point for a representative cell (arrow). The arrowhead shows a cell that remains uninfected over time. Data are representative of at least five experiments.
    Figure Legend Snippet: Kinetics of nuclear localization of GFP after DENV infection. Vero cells transfected with p4B5-EGFP were infected with DENV-2 16681 and sequential images were acquired to assess the time for GFP to localize into the nucleus. Each set of images shows the expression of GFP in p4B5-EGFP transfected cells (top row), nuclear stain using NucBlue (middle row) and transmitted images (bottom row) at each time point. Time after addition of virus to the culture is located at the lower left of each image (magnification, ×20). Nuclear/total fluorescence (N/T) was calculated at each time point for a representative cell (arrow). The arrowhead shows a cell that remains uninfected over time. Data are representative of at least five experiments.

    Techniques Used: Infection, Transfection, Expressing, Staining, Fluorescence

    Nuclear localization of GFP correlates with DENV antigen and co-expression of the NS2B3 protease. (a) Vero cells were transfected with p4B5-EGFP (green) and infected with DENV-2 16681 at an m.o.i of 1. 24 hours post-infection, cells were fixed, permeabilized and stained with antibody against DENV complex. The NS4B5-EGFP panel shows cytoplasmic expression of GFP (right, arrow neighboring cytoplasmic and nuclear expression of GFP (left, arrowhead) (magnification, ×100). DENV Ag panel shows DENV antigen staining in cells infected with DENV. Overlay of GFP and antigen staining (red) shows that nuclear localization of GFP correlates with DENV antigen staining (magnification, ×100). (b and c) Vero cells transfected with the p4B5-EGFP alone (b) or cotransfected with pNS2B3 (c) were analyzed for nuclear localization of GFP at 48hrs post-transfection (magnification, ×100). NS4B5-EGFP panel shows location of GFP within the cells (green). The nucleus panel shows the nucleus stained with NucBlue (blue, Life Technologies). The NS2B3 panel shows indirect antibody staining for NS3 (red). The overlay shows that nuclear GFP expression correlates with NS2B3 expression. Data are representative of at least six (a) and two (b) experiments.
    Figure Legend Snippet: Nuclear localization of GFP correlates with DENV antigen and co-expression of the NS2B3 protease. (a) Vero cells were transfected with p4B5-EGFP (green) and infected with DENV-2 16681 at an m.o.i of 1. 24 hours post-infection, cells were fixed, permeabilized and stained with antibody against DENV complex. The NS4B5-EGFP panel shows cytoplasmic expression of GFP (right, arrow neighboring cytoplasmic and nuclear expression of GFP (left, arrowhead) (magnification, ×100). DENV Ag panel shows DENV antigen staining in cells infected with DENV. Overlay of GFP and antigen staining (red) shows that nuclear localization of GFP correlates with DENV antigen staining (magnification, ×100). (b and c) Vero cells transfected with the p4B5-EGFP alone (b) or cotransfected with pNS2B3 (c) were analyzed for nuclear localization of GFP at 48hrs post-transfection (magnification, ×100). NS4B5-EGFP panel shows location of GFP within the cells (green). The nucleus panel shows the nucleus stained with NucBlue (blue, Life Technologies). The NS2B3 panel shows indirect antibody staining for NS3 (red). The overlay shows that nuclear GFP expression correlates with NS2B3 expression. Data are representative of at least six (a) and two (b) experiments.

    Techniques Used: Expressing, Transfection, Infection, Staining

    All four DENV serotypes induce cleavage of p4B5-EGFP to localize GFP to the nucleus. Vero cells transfected with p4B5-EGFP (green) were infected with each of the four DENV serotypes at an MOI of 1. Cells were fixed, permeabilized and stained for DENV antigen (red) and nuclear DNA (cyan). Cells were analyzed at 24 hours post-infection by fluorescence microscopy. Each row is a representative image of DENV infected cells in bright field (trans) and fluorescence images showing nuclear stain (cyan), DENV antigen stain (red), 4B5-EGFP expression (green). The overlay is a composite of the nucleus, DENV and NS4B-EGFP images. (a) DENV-1 Hawaii, (b) DENV-2 C0112/96, (c) DENV-3 CH53489 and (d) DENV-4 814669. Data are representative of at least four experiments each.
    Figure Legend Snippet: All four DENV serotypes induce cleavage of p4B5-EGFP to localize GFP to the nucleus. Vero cells transfected with p4B5-EGFP (green) were infected with each of the four DENV serotypes at an MOI of 1. Cells were fixed, permeabilized and stained for DENV antigen (red) and nuclear DNA (cyan). Cells were analyzed at 24 hours post-infection by fluorescence microscopy. Each row is a representative image of DENV infected cells in bright field (trans) and fluorescence images showing nuclear stain (cyan), DENV antigen stain (red), 4B5-EGFP expression (green). The overlay is a composite of the nucleus, DENV and NS4B-EGFP images. (a) DENV-1 Hawaii, (b) DENV-2 C0112/96, (c) DENV-3 CH53489 and (d) DENV-4 814669. Data are representative of at least four experiments each.

    Techniques Used: Transfection, Infection, Staining, Fluorescence, Microscopy, Expressing

    Construction and characterization of p4B5-EGFP (a) Schematic of the p4B5-EGFP construct containing NS4B and the first 30 nucleotides of NS5 of the DENV-2 genome tagged with the SV40 NLS and eGFP. The arrow indicates the cleavage site utilized by the DENV NS2B3 protease. The vertical lines represent restriction endonuclease sites. (b) Unfixed Vero cells transfected with p4B5-EGFP were stained with ER-Tracker Blue-White DPX dye (Cyan) 24hrs post-transfection and immediately imaged to detect colocalization of NS4B5-EGFP (reporter, green) with ER membranes (blue, magnification, ×100). Inset reflects ER staining of cells alone. (c) Unfixed Vero cells transfected with p4B5-EGFP were infected with DENV-2 16681. Nuclei were counterstained with NucBlue for live cells (Invitrogen) (blue) to detect nuclear localization of GFP (green, magnification, ×100). Cytoplasmic expression of GFP is indicated by the arrowhead and nuclear expression of GFP is indicated by an arrow (magnification, ×100). (d) Analysis of nuclear to total fluorescence intensity ratios of GFP (nuclear:total fl) in uninfected and DENV infected cells at 24 h was performed using ImageJ software. Each symbol represents analysis of a single cell. Statistical analysis was performed using nonparametric Wilcoxon-Mann-Whitney rank sum test. Straight line represents the median. (e) Cellular lysates were prepared 24 h post infection and subjected to Western blotting for GFP or β-actin. The 5-EGFP fragment represents the cleavage product resulting from cleavage between NS4B and NS5-EGFP during viral infection.
    Figure Legend Snippet: Construction and characterization of p4B5-EGFP (a) Schematic of the p4B5-EGFP construct containing NS4B and the first 30 nucleotides of NS5 of the DENV-2 genome tagged with the SV40 NLS and eGFP. The arrow indicates the cleavage site utilized by the DENV NS2B3 protease. The vertical lines represent restriction endonuclease sites. (b) Unfixed Vero cells transfected with p4B5-EGFP were stained with ER-Tracker Blue-White DPX dye (Cyan) 24hrs post-transfection and immediately imaged to detect colocalization of NS4B5-EGFP (reporter, green) with ER membranes (blue, magnification, ×100). Inset reflects ER staining of cells alone. (c) Unfixed Vero cells transfected with p4B5-EGFP were infected with DENV-2 16681. Nuclei were counterstained with NucBlue for live cells (Invitrogen) (blue) to detect nuclear localization of GFP (green, magnification, ×100). Cytoplasmic expression of GFP is indicated by the arrowhead and nuclear expression of GFP is indicated by an arrow (magnification, ×100). (d) Analysis of nuclear to total fluorescence intensity ratios of GFP (nuclear:total fl) in uninfected and DENV infected cells at 24 h was performed using ImageJ software. Each symbol represents analysis of a single cell. Statistical analysis was performed using nonparametric Wilcoxon-Mann-Whitney rank sum test. Straight line represents the median. (e) Cellular lysates were prepared 24 h post infection and subjected to Western blotting for GFP or β-actin. The 5-EGFP fragment represents the cleavage product resulting from cleavage between NS4B and NS5-EGFP during viral infection.

    Techniques Used: Construct, Transfection, Staining, Infection, Expressing, Fluorescence, Software, MANN-WHITNEY, Western Blot

    24) Product Images from "Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi"

    Article Title: Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00434

    Parasitic load quantification in VERO cells infected with TcNTPDase-1 hemi-knockouts. VERO cells were infected for 48 h with metacyclic trypomastigotes of WT, HYGRO, and NEO hemi-knockout clones. DNA was extracted and parasitic load was estimated by real-time PCR. * p
    Figure Legend Snippet: Parasitic load quantification in VERO cells infected with TcNTPDase-1 hemi-knockouts. VERO cells were infected for 48 h with metacyclic trypomastigotes of WT, HYGRO, and NEO hemi-knockout clones. DNA was extracted and parasitic load was estimated by real-time PCR. * p

    Techniques Used: Infection, Knock-Out, Clone Assay, Real-time Polymerase Chain Reaction

    25) Product Images from "Increased toxin expression in a Clostridium difficile mfd mutant"

    Article Title: Increased toxin expression in a Clostridium difficile mfd mutant

    Journal: BMC Microbiology

    doi: 10.1186/s12866-015-0611-5

    Cytotoxicity levels of toxins A and B in the mfd mutant. Cultures of 630Δ erm, the mfd mutant or the complement were grown in TY or TYG media. Cell lysates and culture supernatants were prepared at various time points. Samples were normalized for optical density of the culture and analyzed for the presence of toxins A and B by cytotoxicity on HT29 cells (toxin A) or Vero cells (toxin B). Panels: a bacterial lysates on Vero cells; b bacterial supernatants on Vero cells; c bacterial lysates on HT29 cells; d bacterial supernatants on HT29 cells. Samples were standardized by OD at point of collection. ○, 630Δ erm; ■ mfd mutant; ▲ complement. Data represents the mean of two biological repeats and at least two technical repeats and error bars represent standard deviations
    Figure Legend Snippet: Cytotoxicity levels of toxins A and B in the mfd mutant. Cultures of 630Δ erm, the mfd mutant or the complement were grown in TY or TYG media. Cell lysates and culture supernatants were prepared at various time points. Samples were normalized for optical density of the culture and analyzed for the presence of toxins A and B by cytotoxicity on HT29 cells (toxin A) or Vero cells (toxin B). Panels: a bacterial lysates on Vero cells; b bacterial supernatants on Vero cells; c bacterial lysates on HT29 cells; d bacterial supernatants on HT29 cells. Samples were standardized by OD at point of collection. ○, 630Δ erm; ■ mfd mutant; ▲ complement. Data represents the mean of two biological repeats and at least two technical repeats and error bars represent standard deviations

    Techniques Used: Mutagenesis

    26) Product Images from "Propolis Extract for Onychomycosis Topical Treatment: From Bench to Clinic"

    Article Title: Propolis Extract for Onychomycosis Topical Treatment: From Bench to Clinic

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00779

    Effect of propolis extract (PE) on HeLa and Vero cell lines. Viability determined by MTT assay. Concentrations of the PE extract ranged from 0.002 to 1.42% of total polyphenols. The results are presented as the average of three independent experiments performed in three replicates, ∗ p
    Figure Legend Snippet: Effect of propolis extract (PE) on HeLa and Vero cell lines. Viability determined by MTT assay. Concentrations of the PE extract ranged from 0.002 to 1.42% of total polyphenols. The results are presented as the average of three independent experiments performed in three replicates, ∗ p

    Techniques Used: MTT Assay

    27) Product Images from "Antiviral and Antioxidant Properties of Echinochrome A"

    Article Title: Antiviral and Antioxidant Properties of Echinochrome A

    Journal: Marine Drugs

    doi: 10.3390/md16120509

    Influence of Ech and studied formulations on the lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formations in Vero cells. The formulations were tested at a concentration of five μg/mL. * p
    Figure Legend Snippet: Influence of Ech and studied formulations on the lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formations in Vero cells. The formulations were tested at a concentration of five μg/mL. * p

    Techniques Used: Concentration Assay

    28) Product Images from "TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells"

    Article Title: TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells

    Journal: Viruses

    doi: 10.3390/v9050114

    Effects of TTSPs and TTSP inhibitor on viral replication. ( A ) Porcine epidemic diarrhea virus (PEDV) titers following the expression of TTSPs in Vero cells. Viral titers were determined by plaque assay. *** p
    Figure Legend Snippet: Effects of TTSPs and TTSP inhibitor on viral replication. ( A ) Porcine epidemic diarrhea virus (PEDV) titers following the expression of TTSPs in Vero cells. Viral titers were determined by plaque assay. *** p

    Techniques Used: Expressing, Plaque Assay

    Culture of PEDV isolated from pig intestine in Vero cells transiently expressing TTSPs in three serial passages. ( A ) Isolation of PEDV strain A; ( B ) Isolation of PEDV strain B. The relative quantity of pCMV-Myc without trypsin at the 1st passage = 1. Error bars indicate standard error of the mean. Results shown are those of a representative experiment performed with triplicate samples.
    Figure Legend Snippet: Culture of PEDV isolated from pig intestine in Vero cells transiently expressing TTSPs in three serial passages. ( A ) Isolation of PEDV strain A; ( B ) Isolation of PEDV strain B. The relative quantity of pCMV-Myc without trypsin at the 1st passage = 1. Error bars indicate standard error of the mean. Results shown are those of a representative experiment performed with triplicate samples.

    Techniques Used: Isolation, Expressing

    Expression of type II transmembrane serine proteases (TTSPs) in transfected Vero cells. ( A ) Post-transfection, the expression of TMPRSS2, HAT, DESC1, and MSPL in transfected Vero cells was detected via indirect immunofluorescence. Bar = 25 μm. Magnification, ×200; ( B ) TTSP expression in transfected Vero cells as determined by western blot. Zymogens and the mature form are indicated; ( C ) TTSPs expression was detected by FACS. The geometric mean channel fluorescence (GMCF) measured in a representative experiment performed with triplicate samples is shown. Error bars indicate standard deviations of three independent experiments.
    Figure Legend Snippet: Expression of type II transmembrane serine proteases (TTSPs) in transfected Vero cells. ( A ) Post-transfection, the expression of TMPRSS2, HAT, DESC1, and MSPL in transfected Vero cells was detected via indirect immunofluorescence. Bar = 25 μm. Magnification, ×200; ( B ) TTSP expression in transfected Vero cells as determined by western blot. Zymogens and the mature form are indicated; ( C ) TTSPs expression was detected by FACS. The geometric mean channel fluorescence (GMCF) measured in a representative experiment performed with triplicate samples is shown. Error bars indicate standard deviations of three independent experiments.

    Techniques Used: Expressing, Transfection, HAT Assay, Immunofluorescence, Western Blot, FACS, Fluorescence

    29) Product Images from "Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition"

    Article Title: Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2009.01385.x

    Effect of cathepsin B and L inhibitors on Ebola VLP entry and EBOV infection of VEROs and DCs VERO (A, C, E) and DCs (B, D) were pretreated with mock and decreasing concentrations of cathepsin B and cathepsin L, respectively. Also, VERO (A) and DCs (B) were mock and VLP treated or EBOV GFP -infected (denoted by bar under graphs). Solid bars represent relative percentage of VEROs (A) and DCs (B) infected by VLPs and white bars represent relative percentage of VEROs (A) and DCs (B) infected by EBOV GFP . Cathepsin B (C, D) and cathepsin L (E) activity was measured in duplicate (solid and white bars) using a fluorogenic substrate and expressed as relative fluorenscence units (RFU) using equivalent amounts of pretreatred VEROs (C, E) and DCs (D). (F) Equivalent amounts of VERO (lane 1) and DC (lane 2) lysates (same lysates used to determine cathepsin activity) were subjected to SDS-PAGE and western blotting using anti-cathepsin L antibody (204106, R D systems, Minneapolis, MN). Arrow denotes positions of Cathepsin L species. (G) VP40+GP VLPs were incubated for 1.5 hours with decreasing concentrations of recombinant cathepsin L (5, 1, 0.2, 0.04 μg/mL) and subjected to western blotting using a polyclonal anti-GP antibody. Arrow denotes positions of GP species.
    Figure Legend Snippet: Effect of cathepsin B and L inhibitors on Ebola VLP entry and EBOV infection of VEROs and DCs VERO (A, C, E) and DCs (B, D) were pretreated with mock and decreasing concentrations of cathepsin B and cathepsin L, respectively. Also, VERO (A) and DCs (B) were mock and VLP treated or EBOV GFP -infected (denoted by bar under graphs). Solid bars represent relative percentage of VEROs (A) and DCs (B) infected by VLPs and white bars represent relative percentage of VEROs (A) and DCs (B) infected by EBOV GFP . Cathepsin B (C, D) and cathepsin L (E) activity was measured in duplicate (solid and white bars) using a fluorogenic substrate and expressed as relative fluorenscence units (RFU) using equivalent amounts of pretreatred VEROs (C, E) and DCs (D). (F) Equivalent amounts of VERO (lane 1) and DC (lane 2) lysates (same lysates used to determine cathepsin activity) were subjected to SDS-PAGE and western blotting using anti-cathepsin L antibody (204106, R D systems, Minneapolis, MN). Arrow denotes positions of Cathepsin L species. (G) VP40+GP VLPs were incubated for 1.5 hours with decreasing concentrations of recombinant cathepsin L (5, 1, 0.2, 0.04 μg/mL) and subjected to western blotting using a polyclonal anti-GP antibody. Arrow denotes positions of GP species.

    Techniques Used: Infection, Activity Assay, SDS Page, Western Blot, Incubation, Recombinant

    30) Product Images from "Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells"

    Article Title: Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056164

    The number of glycosylation sites present on HA determines the virus infection rates in DC-SIGN expressing cells. MDCK (A) and Vero (B) cells, transfected with the DC-SIGN gene (black bars) or not (white bars) were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with A/Netherlands/26/07, A/Netherlands/26/07-Δ125, A/Netherlands/602/09, A/Netherlands/602/09-Δ276 or A/Netherlands/602/09-VN54 N125 N160. The percentage of infected cells relative to the positive control (untreated cells still possessing sialic acid) was assessed after detecting infected cells using a FITC-labeled antibody to the viral nucleoprotein and flow-cytometry.
    Figure Legend Snippet: The number of glycosylation sites present on HA determines the virus infection rates in DC-SIGN expressing cells. MDCK (A) and Vero (B) cells, transfected with the DC-SIGN gene (black bars) or not (white bars) were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with A/Netherlands/26/07, A/Netherlands/26/07-Δ125, A/Netherlands/602/09, A/Netherlands/602/09-Δ276 or A/Netherlands/602/09-VN54 N125 N160. The percentage of infected cells relative to the positive control (untreated cells still possessing sialic acid) was assessed after detecting infected cells using a FITC-labeled antibody to the viral nucleoprotein and flow-cytometry.

    Techniques Used: Infection, Expressing, Transfection, Positive Control, Labeling, Flow Cytometry, Cytometry

    Expression of DC-SIGN supports replication of influenza A viruses in the absence of sialic acids. MDCK (A and C) and Vero cells (B and D) transfected with the DC-SIGN gene (black bars) or not (white bars), were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with five different A/H1N1 viruses (A and B) and four A/H3N2 viruses (C and D). The percentage infected cells relative to the untreated control cells, still possessing sialic acid, was assessed after detecting infected cells using a FITC-labelled antibody to the viral nucleoprotein and flow-cytometry. To confirm that the entry was mediated via DC-SIGN, Vero and Vero DC-SIGN were treated with neuraminidase from vibrio cholerae for 30 minutes to remove sialic acids from the cell surface and incubated with or without antibodies to DC-SIGN or an isotype control antibody as indicated (E and F). These cells were subsequently inoculated with influenza viruses. NL/312/03 and USSR/90/77. The percentage of infected cells compared to the positive control (untreated cells, still possessing sialic acid) was assessed as described above.
    Figure Legend Snippet: Expression of DC-SIGN supports replication of influenza A viruses in the absence of sialic acids. MDCK (A and C) and Vero cells (B and D) transfected with the DC-SIGN gene (black bars) or not (white bars), were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with five different A/H1N1 viruses (A and B) and four A/H3N2 viruses (C and D). The percentage infected cells relative to the untreated control cells, still possessing sialic acid, was assessed after detecting infected cells using a FITC-labelled antibody to the viral nucleoprotein and flow-cytometry. To confirm that the entry was mediated via DC-SIGN, Vero and Vero DC-SIGN were treated with neuraminidase from vibrio cholerae for 30 minutes to remove sialic acids from the cell surface and incubated with or without antibodies to DC-SIGN or an isotype control antibody as indicated (E and F). These cells were subsequently inoculated with influenza viruses. NL/312/03 and USSR/90/77. The percentage of infected cells compared to the positive control (untreated cells, still possessing sialic acid) was assessed as described above.

    Techniques Used: Expressing, Transfection, Infection, Flow Cytometry, Cytometry, Incubation, Positive Control

    DC-SIGN expression in stably transfected MDCK DC-SIGN and Vero DC-SIGN cells. MDCK cells (A) and Vero cells (B) without (dotted line) and transfected with the gene encoding DC-SIGN (solid line) were analyzed for DC-SIGN expression after staining with a PE-labeled antibody to DC-SIGN and flow cytometry.
    Figure Legend Snippet: DC-SIGN expression in stably transfected MDCK DC-SIGN and Vero DC-SIGN cells. MDCK cells (A) and Vero cells (B) without (dotted line) and transfected with the gene encoding DC-SIGN (solid line) were analyzed for DC-SIGN expression after staining with a PE-labeled antibody to DC-SIGN and flow cytometry.

    Techniques Used: Expressing, Stable Transfection, Transfection, Staining, Labeling, Flow Cytometry, Cytometry

    31) Product Images from "Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures"

    Article Title: Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    Journal: Journal of Virology

    doi: 10.1128/JVI.02351-15

    Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product
    Figure Legend Snippet: Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product

    Techniques Used: Expressing, Activity Assay, Infection, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM
    Figure Legend Snippet: Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM

    Techniques Used: Purification

    Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected
    Figure Legend Snippet: Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected

    Techniques Used: Infection, Derivative Assay, Produced, Purification

    Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (
    Figure Legend Snippet: Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (

    Techniques Used: Infection, Purification, Produced

    32) Product Images from "Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication"

    Article Title: Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.80.8.4147-4156.2006

    Diagrammatic representation of the length of each cell cycle stage in asynchronously cycling Vero cells. G 1 phase (white) takes approximately 12 h), S phase (gray) takes approximately 6 h, and G 2 and M phases (black) take approximately 2 and 1 h to complete, respectively. The points in the cell cycle where serum starvation (S), the double-thymidine block (T), and nocodazole (N) block the cell cycle are indicated. On the outside is indicated where in the cell cycle synchronously replicating cells reach when released from their appropriate block.
    Figure Legend Snippet: Diagrammatic representation of the length of each cell cycle stage in asynchronously cycling Vero cells. G 1 phase (white) takes approximately 12 h), S phase (gray) takes approximately 6 h, and G 2 and M phases (black) take approximately 2 and 1 h to complete, respectively. The points in the cell cycle where serum starvation (S), the double-thymidine block (T), and nocodazole (N) block the cell cycle are indicated. On the outside is indicated where in the cell cycle synchronously replicating cells reach when released from their appropriate block.

    Techniques Used: Blocking Assay

    33) Product Images from "Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, which Demonstrates Attenuation and Immunogenicity in Mice"

    Article Title: Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, which Demonstrates Attenuation and Immunogenicity in Mice

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.14-0427

    Sensitivity of Japanese encephalitis virus (JEV) strains to type I interferon (IFN)-treatment of Vero cells. Vero cells were infected at an multiplicity of infection (MOI) of 0.01 PFU/cell, followed by addition of medium-only (untreated) or medium containing
    Figure Legend Snippet: Sensitivity of Japanese encephalitis virus (JEV) strains to type I interferon (IFN)-treatment of Vero cells. Vero cells were infected at an multiplicity of infection (MOI) of 0.01 PFU/cell, followed by addition of medium-only (untreated) or medium containing

    Techniques Used: Infection

    34) Product Images from "Ribavirin efficiently suppresses porcine nidovirus replication"

    Article Title: Ribavirin efficiently suppresses porcine nidovirus replication

    Journal: Virus Research

    doi: 10.1016/j.virusres.2012.10.018

    Inhibition of viral protein translation by ribavirin. Ribavirin-treated PAM-pCD163 and Vero cells were mock-infected or infected with PRRSV (A) or PEDV (B) for 1 h and were further cultivated in the presence or absence of ribavirin. At 48 hpi, cellular lysates were collected, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted by using the antibody that recognizes the PRRSV N protein or the PEDV S protein. The blot was also reacted with mouse MAb against β-actin to verify equal protein loading. Each viral protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the β-actin gene and ribavirin-treated sample results were compared to vehicle-control results. Values are representative of the mean from three independent experiments and error bars denote standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Inhibition of viral protein translation by ribavirin. Ribavirin-treated PAM-pCD163 and Vero cells were mock-infected or infected with PRRSV (A) or PEDV (B) for 1 h and were further cultivated in the presence or absence of ribavirin. At 48 hpi, cellular lysates were collected, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted by using the antibody that recognizes the PRRSV N protein or the PEDV S protein. The blot was also reacted with mouse MAb against β-actin to verify equal protein loading. Each viral protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the β-actin gene and ribavirin-treated sample results were compared to vehicle-control results. Values are representative of the mean from three independent experiments and error bars denote standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Inhibition, Infection, SDS Page, Expressing

    Suppression of nidovirus propagation by ribavirin at early times post-infection. PAM-pCD163 and Vero cells were pretreated with ribavirin and were infected with PRRSV (A) or PEDV (B), respectively. At the indicated times at post-infection, ribavirin was added to achieve a final concentration. At 48 hpi, virus-infected cells were fixed and virus infectivity was determined by quantifying the number of cells expressing N proteins through IFA. Data are representative of the mean of three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Suppression of nidovirus propagation by ribavirin at early times post-infection. PAM-pCD163 and Vero cells were pretreated with ribavirin and were infected with PRRSV (A) or PEDV (B), respectively. At the indicated times at post-infection, ribavirin was added to achieve a final concentration. At 48 hpi, virus-infected cells were fixed and virus infectivity was determined by quantifying the number of cells expressing N proteins through IFA. Data are representative of the mean of three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Infection, Concentration Assay, Expressing, Immunofluorescence

    Reduction of viral progeny production by ribavirin. (A) PAM-pCD163 and Vero cells were pretreated with ribavirin for 1 h and were mock or virus infected (MOI of 0.1). Ribavirin was present in the medium throughout the infection. At 48 hpi, the virus supernatants were collected and the titers of PRRSV (left) and PEDV (right) were determined. (B) Growth kinetics of PRRSV (left) and PEDV (right) upon treatment with ribavirin was assessed exactly as for panel A. At the indicated time points post-infection, culture supernatants were harvested and virus titers were measured. (C) Virus internalization assay. PAM-pCD163 and Vero cells were infected at an MOI of 0.1 at 4 °C for 1 h. After washing with cold PBS, infected cells were maintained in the presence or absence of ribavirin either at 4 °C or 37 °C for an additional hour. Bound but uninternalized virus particles were removed by treatment with protease K. The infected cells were then serially diluted and plated onto fresh target cells. At 2 days post-incubation, internalized viruses were titrated by IFA and plaque assay for PRRSV (left) and PEDV (right), respectively. Results are expressed as the mean values from triplicate wells and error bars represent standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Reduction of viral progeny production by ribavirin. (A) PAM-pCD163 and Vero cells were pretreated with ribavirin for 1 h and were mock or virus infected (MOI of 0.1). Ribavirin was present in the medium throughout the infection. At 48 hpi, the virus supernatants were collected and the titers of PRRSV (left) and PEDV (right) were determined. (B) Growth kinetics of PRRSV (left) and PEDV (right) upon treatment with ribavirin was assessed exactly as for panel A. At the indicated time points post-infection, culture supernatants were harvested and virus titers were measured. (C) Virus internalization assay. PAM-pCD163 and Vero cells were infected at an MOI of 0.1 at 4 °C for 1 h. After washing with cold PBS, infected cells were maintained in the presence or absence of ribavirin either at 4 °C or 37 °C for an additional hour. Bound but uninternalized virus particles were removed by treatment with protease K. The infected cells were then serially diluted and plated onto fresh target cells. At 2 days post-incubation, internalized viruses were titrated by IFA and plaque assay for PRRSV (left) and PEDV (right), respectively. Results are expressed as the mean values from triplicate wells and error bars represent standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Infection, Incubation, Immunofluorescence, Plaque Assay

    Effect of ribavirin on the replication of porcine nidoviruses. PAM-pCD163 and Vero cells were preincubated with ribavirin at indicated concentrations for 1 h prior to infection and were mock-infected or infected with PRRSV (A) or PEDV (B) at an MOI of 0.1. Virus-infected cells were further maintained for 48 h in the presence of vehicle or ribavirin. Virus-specific CPEs were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 100× (first panels). For immunostaining, infected cells were fixed at 48 hpi and incubated with MAb against the N protein of PRRSV or PEDV followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200× magnification. Viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing N proteins through IFA. Five fields at 200× magnification were counted per each condition and the total number of cells per field as determined by DAPI staining was similar in all fields. Values are representative of the mean of three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Effect of ribavirin on the replication of porcine nidoviruses. PAM-pCD163 and Vero cells were preincubated with ribavirin at indicated concentrations for 1 h prior to infection and were mock-infected or infected with PRRSV (A) or PEDV (B) at an MOI of 0.1. Virus-infected cells were further maintained for 48 h in the presence of vehicle or ribavirin. Virus-specific CPEs were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 100× (first panels). For immunostaining, infected cells were fixed at 48 hpi and incubated with MAb against the N protein of PRRSV or PEDV followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200× magnification. Viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing N proteins through IFA. Five fields at 200× magnification were counted per each condition and the total number of cells per field as determined by DAPI staining was similar in all fields. Values are representative of the mean of three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Infection, Inverted Microscopy, Immunostaining, Incubation, Microscopy, Expressing, Immunofluorescence, Staining

    Inhibition of viral RNA transcription by ribavirin. PAM-pCD163 and Vero cells pretreated with ribavirin were mock-infected or infected with PRRSV (A) or PEDV (B) for 1 h and were incubated in the presence of ribavirin. Total cellular RNA was extracted at 48 hpi, and strand-specific viral genomic RNAs (black bars) and sg mRNAs (white bars) of PRRSV and PEDV were amplified by quantitative real-time RT-PCR. Viral positive-sense genomic RNA and sg mRNA were normalized to mRNA for porcine β-actin or monkey GAPDH and relative quantities (RQ) of mRNA accumulation were evaluated. Ribavirin-treated sample results were compared with untreated results. Values are representative of the mean from three independent experiments and error bars denote standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Inhibition of viral RNA transcription by ribavirin. PAM-pCD163 and Vero cells pretreated with ribavirin were mock-infected or infected with PRRSV (A) or PEDV (B) for 1 h and were incubated in the presence of ribavirin. Total cellular RNA was extracted at 48 hpi, and strand-specific viral genomic RNAs (black bars) and sg mRNAs (white bars) of PRRSV and PEDV were amplified by quantitative real-time RT-PCR. Viral positive-sense genomic RNA and sg mRNA were normalized to mRNA for porcine β-actin or monkey GAPDH and relative quantities (RQ) of mRNA accumulation were evaluated. Ribavirin-treated sample results were compared with untreated results. Values are representative of the mean from three independent experiments and error bars denote standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Inhibition, Infection, Incubation, Amplification, Quantitative RT-PCR

    Effects of supplementation of guanosine in ribavirin treatment and MPA on porcine nidovirus infection. (A) Effect of ribavirin alone or with guanosine (100 μM) on the replication of PRRSV (left) and PEDV (right). PAM-pCD163 and Vero cells were preincubated with various concentrations of ribavirin with (+) or without 100 (−) μM guanosine. Virus-infected cells were fixed at 48 hpi and subjected to immunofluorescence assay. Viral productions were measured by quantifying the number of cells expressing N proteins through IFA as described above. Viral productions in cells treated by guanosine and ribavirin that were significantly different from those in cells treated with ribavirin alone are indicated. (B) Effect of MPA on the replication of PRRSV (left) and PEDV (right). PAM-pCD163 and Vero cells were preincubated with various concentrations of MPA ranging from 0 (mock) to 10 μM and infected with PRRSV or PEDV. At 48 hpi, virus-infected cells were subjected to immunofluorescence assay, and viral productions were measured by quantifying the number of cells expressing N proteins through IFA as described above. Data are representative of the mean values from three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P
    Figure Legend Snippet: Effects of supplementation of guanosine in ribavirin treatment and MPA on porcine nidovirus infection. (A) Effect of ribavirin alone or with guanosine (100 μM) on the replication of PRRSV (left) and PEDV (right). PAM-pCD163 and Vero cells were preincubated with various concentrations of ribavirin with (+) or without 100 (−) μM guanosine. Virus-infected cells were fixed at 48 hpi and subjected to immunofluorescence assay. Viral productions were measured by quantifying the number of cells expressing N proteins through IFA as described above. Viral productions in cells treated by guanosine and ribavirin that were significantly different from those in cells treated with ribavirin alone are indicated. (B) Effect of MPA on the replication of PRRSV (left) and PEDV (right). PAM-pCD163 and Vero cells were preincubated with various concentrations of MPA ranging from 0 (mock) to 10 μM and infected with PRRSV or PEDV. At 48 hpi, virus-infected cells were subjected to immunofluorescence assay, and viral productions were measured by quantifying the number of cells expressing N proteins through IFA as described above. Data are representative of the mean values from three independent experiments and error bars represent standard deviations. * P = 0.001–0.05; † P

    Techniques Used: Infection, Immunofluorescence, Expressing

    35) Product Images from "Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters"

    Article Title: Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

    Journal: Journal of Virology

    doi: 10.1128/JVI.02632-12

    Infection with the recombinant MV with the F protein lacking the cytoplasmic tail (IC323-EGFP-FΔ30). SLAM- and nectin 4-negative Vero (A) and IMR-32 (B) cells were infected with IC323-EGFP or IC323-EGFP-FΔ30 at an MOI of 0.1. At 72 h after
    Figure Legend Snippet: Infection with the recombinant MV with the F protein lacking the cytoplasmic tail (IC323-EGFP-FΔ30). SLAM- and nectin 4-negative Vero (A) and IMR-32 (B) cells were infected with IC323-EGFP or IC323-EGFP-FΔ30 at an MOI of 0.1. At 72 h after

    Techniques Used: Infection, Recombinant

    Amino acid substitutions in the F protein enhance cell-cell fusion in Vero/hSLAM and Vero cells. (A) Vero/hSLAM cells were transfected with expression plasmids encoding wild-type (Wt) or mutant F proteins with the indicated substitutions, together with
    Figure Legend Snippet: Amino acid substitutions in the F protein enhance cell-cell fusion in Vero/hSLAM and Vero cells. (A) Vero/hSLAM cells were transfected with expression plasmids encoding wild-type (Wt) or mutant F proteins with the indicated substitutions, together with

    Techniques Used: Transfection, Expressing, Mutagenesis

    Syncytium formation in SLAM- and nectin 4-negative cells infected with recombinant MVs. Vero (A), IMR-32 (B), and SYM-1 (C) cells were infected with the parental recombinant MV expressing EGFP (IC323-EGFP) or its mutants expressing the F protein with
    Figure Legend Snippet: Syncytium formation in SLAM- and nectin 4-negative cells infected with recombinant MVs. Vero (A), IMR-32 (B), and SYM-1 (C) cells were infected with the parental recombinant MV expressing EGFP (IC323-EGFP) or its mutants expressing the F protein with

    Techniques Used: Infection, Recombinant, Expressing

    36) Product Images from "Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment"

    Article Title: Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00074

    HSV-1 infection of the ENS. (A) The content of the ileum was collected from mice at 15 and 45 min post-IG inoculum with HSV-1 strain 16 (1 × 10 7 PFU). Intestinal content was co-cultured on Vero cells and plaques were counted. The number of plaques derived from Vero cells incubated with HSV-1 strain SC16 (1 × 10 7 PFU) but not injected in mice was taken as 100%. Data are represented as mean ± SEM of three separated experiments. n = 4 mice per group. (B) Total DNA was isolated from Vero cells exposed for up to 72 h to content of ileum obtained from mice inoculated IG with HSV-1 or sham infected. DNA was subjected to PCR using HSV-1 tk specific primers. Representative picture of one out of three agarose gel is reported. bp: base pairs. (C) Primary enteric neurons were infected with HSV-1, fixed 36 h later, and probed with anti-β-III-tubulin (red) and anti-ICP-27 (green) antibodies. Cells were imaged using a confocal microscope. Representative images are shown. (D) One, two, and three weeks post-IG inoculum of HSV-1 strain SC16, total RNA was purified from LMMP and semi-quantitative PCR was performed to evaluate the expression of HSV-1 LATs and early (ICP4, tk) mRNA transcripts. A representative experiment is shown. Rn18S was used as extraction and loading control. Sham: mice IG inoculated with Vero cell lysate.
    Figure Legend Snippet: HSV-1 infection of the ENS. (A) The content of the ileum was collected from mice at 15 and 45 min post-IG inoculum with HSV-1 strain 16 (1 × 10 7 PFU). Intestinal content was co-cultured on Vero cells and plaques were counted. The number of plaques derived from Vero cells incubated with HSV-1 strain SC16 (1 × 10 7 PFU) but not injected in mice was taken as 100%. Data are represented as mean ± SEM of three separated experiments. n = 4 mice per group. (B) Total DNA was isolated from Vero cells exposed for up to 72 h to content of ileum obtained from mice inoculated IG with HSV-1 or sham infected. DNA was subjected to PCR using HSV-1 tk specific primers. Representative picture of one out of three agarose gel is reported. bp: base pairs. (C) Primary enteric neurons were infected with HSV-1, fixed 36 h later, and probed with anti-β-III-tubulin (red) and anti-ICP-27 (green) antibodies. Cells were imaged using a confocal microscope. Representative images are shown. (D) One, two, and three weeks post-IG inoculum of HSV-1 strain SC16, total RNA was purified from LMMP and semi-quantitative PCR was performed to evaluate the expression of HSV-1 LATs and early (ICP4, tk) mRNA transcripts. A representative experiment is shown. Rn18S was used as extraction and loading control. Sham: mice IG inoculated with Vero cell lysate.

    Techniques Used: Infection, Mouse Assay, Cell Culture, Derivative Assay, Incubation, Injection, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Microscopy, Purification, Real-time Polymerase Chain Reaction, Expressing

    37) Product Images from "Protein encoded by HSV‐1 stimulation‐related gene 1 (HSRG1) interacts with and inhibits SV40 large T antigen"

    Article Title: Protein encoded by HSV‐1 stimulation‐related gene 1 (HSRG1) interacts with and inhibits SV40 large T antigen

    Journal: Cell Proliferation

    doi: 10.1111/j.1365-2184.2006.00408.x

    Expression of HSRG1 impairs the ability of the large T protein to affect the cell cycle and HSRG1 inhibits the transforming ability of SV40 large T protein in vivo . (a) KMB‐17 cells were transfected with pcDNA‐T, pcDNA‐hsrg1 or pcDNA3.0. The samples were collected and assayed by flow cytometry at 12, 24, 36, 48, 60 and 72 h. (b) Vero cells that expressed HSRG1 protein stably were transfected with pcDNA‐LT and pcDNA, respectively. After 36 h of transfection, the cells were trypsinized and diluted to 100, 500, 1000 and 1500 cells/ml in DMEM for subculturing in 6‐well plates according to standard protocols. The colonies formed in agar media were counted after 10 days. Data were collected from experiments and were repeated three times.
    Figure Legend Snippet: Expression of HSRG1 impairs the ability of the large T protein to affect the cell cycle and HSRG1 inhibits the transforming ability of SV40 large T protein in vivo . (a) KMB‐17 cells were transfected with pcDNA‐T, pcDNA‐hsrg1 or pcDNA3.0. The samples were collected and assayed by flow cytometry at 12, 24, 36, 48, 60 and 72 h. (b) Vero cells that expressed HSRG1 protein stably were transfected with pcDNA‐LT and pcDNA, respectively. After 36 h of transfection, the cells were trypsinized and diluted to 100, 500, 1000 and 1500 cells/ml in DMEM for subculturing in 6‐well plates according to standard protocols. The colonies formed in agar media were counted after 10 days. Data were collected from experiments and were repeated three times.

    Techniques Used: Expressing, In Vivo, Transfection, Flow Cytometry, Stable Transfection, Subculturing Assay

    38) Product Images from "TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells"

    Article Title: TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells

    Journal: Viruses

    doi: 10.3390/v9050114

    Effects of TTSPs and TTSP inhibitor on viral replication. ( A ) Porcine epidemic diarrhea virus (PEDV) titers following the expression of TTSPs in Vero cells. Viral titers were determined by plaque assay. *** p
    Figure Legend Snippet: Effects of TTSPs and TTSP inhibitor on viral replication. ( A ) Porcine epidemic diarrhea virus (PEDV) titers following the expression of TTSPs in Vero cells. Viral titers were determined by plaque assay. *** p

    Techniques Used: Expressing, Plaque Assay

    Expression of type II transmembrane serine proteases (TTSPs) in transfected Vero cells. ( A ) Post-transfection, the expression of TMPRSS2, HAT, DESC1, and MSPL in transfected Vero cells was detected via indirect immunofluorescence. Bar = 25 μm. Magnification, ×200; ( B ) TTSP expression in transfected Vero cells as determined by western blot. Zymogens and the mature form are indicated; ( C ) TTSPs expression was detected by FACS. The geometric mean channel fluorescence (GMCF) measured in a representative experiment performed with triplicate samples is shown. Error bars indicate standard deviations of three independent experiments.
    Figure Legend Snippet: Expression of type II transmembrane serine proteases (TTSPs) in transfected Vero cells. ( A ) Post-transfection, the expression of TMPRSS2, HAT, DESC1, and MSPL in transfected Vero cells was detected via indirect immunofluorescence. Bar = 25 μm. Magnification, ×200; ( B ) TTSP expression in transfected Vero cells as determined by western blot. Zymogens and the mature form are indicated; ( C ) TTSPs expression was detected by FACS. The geometric mean channel fluorescence (GMCF) measured in a representative experiment performed with triplicate samples is shown. Error bars indicate standard deviations of three independent experiments.

    Techniques Used: Expressing, Transfection, HAT Assay, Immunofluorescence, Western Blot, FACS, Fluorescence

    39) Product Images from "A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region ▿"

    Article Title: A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05554-11

    The G-deficient rBDV establishes persistent infection in cultured cells. (A) Growth kinetics of ΔGLLP/M-GFP virus in Vero-BG and Vero cells. The cell-free virus was inoculated onto the cells at an MOI of 0.01. Infected cells were split every 3
    Figure Legend Snippet: The G-deficient rBDV establishes persistent infection in cultured cells. (A) Growth kinetics of ΔGLLP/M-GFP virus in Vero-BG and Vero cells. The cell-free virus was inoculated onto the cells at an MOI of 0.01. Infected cells were split every 3

    Techniques Used: Infection, Cell Culture

    40) Product Images from "Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles"

    Article Title: Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1093/ecam/nep157

    The morphology of Vero cells without treatment (a) and with treatment of ACV (b) or excoecarianin (c)-(d). Vero cells were seeded onto 24-well culture plates at density of 1 × 10 3 cells per well. After 4 h, excoecarianin or ACV was added. The cells were incubated for 7 days, and the cellular morphology was examined under phase-contrast microscope.
    Figure Legend Snippet: The morphology of Vero cells without treatment (a) and with treatment of ACV (b) or excoecarianin (c)-(d). Vero cells were seeded onto 24-well culture plates at density of 1 × 10 3 cells per well. After 4 h, excoecarianin or ACV was added. The cells were incubated for 7 days, and the cellular morphology was examined under phase-contrast microscope.

    Techniques Used: Incubation, Microscopy

    Inhibitory effect of excoecarianin (open bars) and ACV (dotted bars) against HSV-2 infection in Vero cell as determined by plaque reduction assay. Vero cells were incubated with 100 pfu of HSV-2 and different concentrations of excoecarianin or ACV. After 1 h, an overlay medium containing 1% methylcellulose was added. On Day 3 post-infection, the cell monolayer was stained with crystal violet and the virus plaques formed were counted. The percentage of inhibition was calculated by comparing the plaque number of compound-treated group to that of the untreated group. The concentrations of excoecarianin and ACV that inhibited 50% of HSV-2 infection (IC 50 ) were determined. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control ( P
    Figure Legend Snippet: Inhibitory effect of excoecarianin (open bars) and ACV (dotted bars) against HSV-2 infection in Vero cell as determined by plaque reduction assay. Vero cells were incubated with 100 pfu of HSV-2 and different concentrations of excoecarianin or ACV. After 1 h, an overlay medium containing 1% methylcellulose was added. On Day 3 post-infection, the cell monolayer was stained with crystal violet and the virus plaques formed were counted. The percentage of inhibition was calculated by comparing the plaque number of compound-treated group to that of the untreated group. The concentrations of excoecarianin and ACV that inhibited 50% of HSV-2 infection (IC 50 ) were determined. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control ( P

    Techniques Used: Infection, Incubation, Staining, Inhibition

    Synergistic antiviral activity of ACV and excoecarianin in Vero cells. IC 50 values were derived from the data shown in Table 3 and used to construct the isobologram. FIC 50 of ACV represents the ratio of the IC 50 of ACV in the presence of a constant concentration of excoecarianin to the IC 50 of ACV alone. The x -axis represents the ratio of the fixed concentration of excoecarianin to the IC 50 of excoecarianin alone. In this representation, displacement of the experimental data points to the left of the theoretical line is indicative of synergistic behavior.
    Figure Legend Snippet: Synergistic antiviral activity of ACV and excoecarianin in Vero cells. IC 50 values were derived from the data shown in Table 3 and used to construct the isobologram. FIC 50 of ACV represents the ratio of the IC 50 of ACV in the presence of a constant concentration of excoecarianin to the IC 50 of ACV alone. The x -axis represents the ratio of the fixed concentration of excoecarianin to the IC 50 of excoecarianin alone. In this representation, displacement of the experimental data points to the left of the theoretical line is indicative of synergistic behavior.

    Techniques Used: Activity Assay, Derivative Assay, Construct, Concentration Assay

    The cytotoxic effect of excoecarianin (open bars) and ACV (dotted bars) toward Vero cells as determined by XTT assay. Various concentrations of excoecarianin or ACV were added to Vero cells. After 72 h of incubation, the XTT solution was added and then the optical densities were measured. The cytotoxic effect of excoecarianin and ACV were evaluated and the 50% cytotoxic concentration (CC 50 ) was calculated. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control ( P
    Figure Legend Snippet: The cytotoxic effect of excoecarianin (open bars) and ACV (dotted bars) toward Vero cells as determined by XTT assay. Various concentrations of excoecarianin or ACV were added to Vero cells. After 72 h of incubation, the XTT solution was added and then the optical densities were measured. The cytotoxic effect of excoecarianin and ACV were evaluated and the 50% cytotoxic concentration (CC 50 ) was calculated. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control ( P

    Techniques Used: XTT Assay, Incubation, Concentration Assay

    Related Articles

    MTT Assay:

    Article Title: Evaluation of the Antiviral Potential of Halogenated Dihydrorugosaflavonoids and Molecular Modeling with nsP3 Protein of Chikungunya Virus (CHIKV)
    Article Snippet: .. Cytotoxicity Studies The cytotoxicity of 5c and 5d compounds for Vero cells was measured using MTT (Sigma) in a 96-well plate format. .. Stocks of 5c and 5d compounds were prepared by dissolving in dimethyl sulfoxide (DMSO), and the working concentration was prepared by further diluting in culture media.

    Cell Culture:

    Article Title: Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner
    Article Snippet: .. Inhibitor treatment Vero cells or Vero cells expressing eGFP-Rab7 cultured in 35 mm glass-bottom culture dishes were pretreated with 100 µM dynasore (Sigma-Aldrich), 2 µM cytochalasin D (Sigma-Aldrich), 50 µM LY294002 hydrochloride (Sigma-Aldrich), 50 nM wortmannin (Sigma-Aldrich), 100 µM EIPA (Sigma-Aldrich) or 100 nM staurosporine (Sigma-Aldrich) for 30 min at 37°C. .. DiI-labeled virions were adsorbed to the cells for 30 min on ice in the presence of these inhibitors in phenol red-free MEM (Invitrogen) containing 2% FBS and 4% BSA.

    Incubation:

    Article Title: Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, which Demonstrates Attenuation and Immunogenicity in Mice
    Article Snippet: .. Virus was added to Vero cells in 24-well plates at an MOI of 0.01 PFU/cell and incubated for 1 hr followed by addition of media-only or media containing 10 U/mL of IFN-α A/D (Sigma-Aldrich), in duplicate per experiment. .. Virus dilutions used for infection were immediately back titrated in Vero cells to ensure consistent doses/well.

    Infection:

    Article Title: A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes
    Article Snippet: .. One 25 cm2 flask of DENV-2 infected Vero cells was also used to prepare an infected cell lysate for the Western blot assays by discarding the supernatant and repeatedly washing the cells with RPMI medium before the addition of 2 ml of 32 mM orthophosphoric acid/58 mM Tris base (pH 6.7) (345245/T6066: Sigma-Aldrich, USA) containing 10% sodium dodecyl sulphate (SDS) (L3771, Sigma, USA) (cell-lysis buffer). ..

    Expressing:

    Article Title: Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner
    Article Snippet: .. Inhibitor treatment Vero cells or Vero cells expressing eGFP-Rab7 cultured in 35 mm glass-bottom culture dishes were pretreated with 100 µM dynasore (Sigma-Aldrich), 2 µM cytochalasin D (Sigma-Aldrich), 50 µM LY294002 hydrochloride (Sigma-Aldrich), 50 nM wortmannin (Sigma-Aldrich), 100 µM EIPA (Sigma-Aldrich) or 100 nM staurosporine (Sigma-Aldrich) for 30 min at 37°C. .. DiI-labeled virions were adsorbed to the cells for 30 min on ice in the presence of these inhibitors in phenol red-free MEM (Invitrogen) containing 2% FBS and 4% BSA.

    Article Title: Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells
    Article Snippet: .. Vero cells obtained from the ATCC and Vero cells expressing TMPRSS2 (Vero/TMPRSS2) ( ) were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, USA) supplemented with 5% heat-inactivated fetal bovine serum (Gibco-BRL, USA). ..

    Modification:

    Article Title: Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells
    Article Snippet: .. Vero cells obtained from the ATCC and Vero cells expressing TMPRSS2 (Vero/TMPRSS2) ( ) were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, USA) supplemented with 5% heat-inactivated fetal bovine serum (Gibco-BRL, USA). ..

    Western Blot:

    Article Title: A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes
    Article Snippet: .. One 25 cm2 flask of DENV-2 infected Vero cells was also used to prepare an infected cell lysate for the Western blot assays by discarding the supernatant and repeatedly washing the cells with RPMI medium before the addition of 2 ml of 32 mM orthophosphoric acid/58 mM Tris base (pH 6.7) (345245/T6066: Sigma-Aldrich, USA) containing 10% sodium dodecyl sulphate (SDS) (L3771, Sigma, USA) (cell-lysis buffer). ..

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  • 99
    Millipore vero cells
    Onset of HSV-2 growth is delayed by PD98059. <t>Vero</t> cells were pretreated (1 h, <t>37°C)</t> with 50 μM PD98059 (○) or PD98059 reconstitution medium (●) and infected with HSV-2. They were reincubated in medium containing 1% FCS with or without PD98059, respectively. Adsorption was for 1 h at 37°C (0 h of growth curve). Virus titers were determined at 2 to 48 h after adsorption, and the results are expressed as the burst size (PFU/cell ± the standard error of the mean). Cells infected with ICP10ΔPK in medium containing 10% FCS (▵) were studied in parallel.
    Vero Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 14 article reviews
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    88
    Millipore sars cov infected vero e6 cells
    SNAP treatment causes a reduction in the production of positive-stranded viral RNA. <t>Vero</t> <t>E6</t> cells were either treated with 400 μM SNAP, NAP or mock-treated and infected with <t>SARS-CoV</t> at an MOI of 1. A parallel set of cells were additionally treated an hour before infection. Cells were harvested from all sets of treatments at 3 h and 24 h post infection and subjected to reverse transcription and realtime PCR using primers and probes specific for the N gene of SARS-CoV and GAPDH. Ct values obtained for N in each sample was normalized against GAPDH control values and plotted for each time point and treatment.
    Sars Cov Infected Vero E6 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore vero e6 cells
    Lysotracker Red-DND99 labels LAMP1 positive lysosomes in HeLa-mCC1a and <t>Vero</t> E6 cells. Scale bar 1μm.
    Vero E6 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Onset of HSV-2 growth is delayed by PD98059. Vero cells were pretreated (1 h, 37°C) with 50 μM PD98059 (○) or PD98059 reconstitution medium (●) and infected with HSV-2. They were reincubated in medium containing 1% FCS with or without PD98059, respectively. Adsorption was for 1 h at 37°C (0 h of growth curve). Virus titers were determined at 2 to 48 h after adsorption, and the results are expressed as the burst size (PFU/cell ± the standard error of the mean). Cells infected with ICP10ΔPK in medium containing 10% FCS (▵) were studied in parallel.

    Journal: Journal of Virology

    Article Title: Ras-GAP Binding and Phosphorylation by Herpes Simplex Virus Type 2 RR1 PK (ICP10) and Activation of the Ras/MEK/MAPK Mitogenic Pathway Are Required for Timely Onset of Virus Growth

    doi:

    Figure Lengend Snippet: Onset of HSV-2 growth is delayed by PD98059. Vero cells were pretreated (1 h, 37°C) with 50 μM PD98059 (○) or PD98059 reconstitution medium (●) and infected with HSV-2. They were reincubated in medium containing 1% FCS with or without PD98059, respectively. Adsorption was for 1 h at 37°C (0 h of growth curve). Virus titers were determined at 2 to 48 h after adsorption, and the results are expressed as the burst size (PFU/cell ± the standard error of the mean). Cells infected with ICP10ΔPK in medium containing 10% FCS (▵) were studied in parallel.

    Article Snippet: Vero cells were pretreated (1 h, 37°C) with 50 μM MEK inhibitor PD98059 (Calbiochem, LaJolla, Calif.) or were mock treated with reconstitution solution free of PD98059 and then infected with HSV-2 in the presence or absence of PD98059.

    Techniques: Infection, Adsorption

    SNAP treatment causes a reduction in the production of positive-stranded viral RNA. Vero E6 cells were either treated with 400 μM SNAP, NAP or mock-treated and infected with SARS-CoV at an MOI of 1. A parallel set of cells were additionally treated an hour before infection. Cells were harvested from all sets of treatments at 3 h and 24 h post infection and subjected to reverse transcription and realtime PCR using primers and probes specific for the N gene of SARS-CoV and GAPDH. Ct values obtained for N in each sample was normalized against GAPDH control values and plotted for each time point and treatment.

    Journal: Virology

    Article Title: Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

    doi: 10.1016/j.virol.2009.09.007

    Figure Lengend Snippet: SNAP treatment causes a reduction in the production of positive-stranded viral RNA. Vero E6 cells were either treated with 400 μM SNAP, NAP or mock-treated and infected with SARS-CoV at an MOI of 1. A parallel set of cells were additionally treated an hour before infection. Cells were harvested from all sets of treatments at 3 h and 24 h post infection and subjected to reverse transcription and realtime PCR using primers and probes specific for the N gene of SARS-CoV and GAPDH. Ct values obtained for N in each sample was normalized against GAPDH control values and plotted for each time point and treatment.

    Article Snippet: Similarly, SARS-CoV infected Vero E6 cells were treated with a superoxide scavenger, Mn(III)tetrakis(4-Benzoic acid)porphyrin chloride (MnTBAP; Calbiochem), in the absence or presence of 400 μM SIN-1.

    Techniques: Infection, Polymerase Chain Reaction

    SNAP treatment causes a difference in observed levels of replicase polyprotein cleavage products. Lysates from either uninfected or SARS-CoV infected Vero E6 cells which had either been treated with SNAP, NAP or mock-treated harvested 24 h post-infection were subjected to Western blot analysis using a monoclonal antibody targeting the nsp8 protein.

    Journal: Virology

    Article Title: Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

    doi: 10.1016/j.virol.2009.09.007

    Figure Lengend Snippet: SNAP treatment causes a difference in observed levels of replicase polyprotein cleavage products. Lysates from either uninfected or SARS-CoV infected Vero E6 cells which had either been treated with SNAP, NAP or mock-treated harvested 24 h post-infection were subjected to Western blot analysis using a monoclonal antibody targeting the nsp8 protein.

    Article Snippet: Similarly, SARS-CoV infected Vero E6 cells were treated with a superoxide scavenger, Mn(III)tetrakis(4-Benzoic acid)porphyrin chloride (MnTBAP; Calbiochem), in the absence or presence of 400 μM SIN-1.

    Techniques: Infection, Western Blot

    SIN-1 treatment has no antiviral effect on SARS-CoV infected Vero E6 cells. Vero E6 cells were infected with SARS-CoV at an MOI of 1.0, at 1 hpi cells were treated with different concentrations of SIN-1 and/or MnTBAP. (A) Cells treated with different concentrations of SIN-1. 24 hpi, virus was harvested and titers determined. (B) Cells treated with 400 μM SIN-1 and different concentrations of MnTBAP. Virus was harvested 24 hpi and titers determined. (C) Cells treated with different concentrations of MnTBAP, virus was harvested 24 hpi and titers determined.

    Journal: Virology

    Article Title: Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

    doi: 10.1016/j.virol.2009.09.007

    Figure Lengend Snippet: SIN-1 treatment has no antiviral effect on SARS-CoV infected Vero E6 cells. Vero E6 cells were infected with SARS-CoV at an MOI of 1.0, at 1 hpi cells were treated with different concentrations of SIN-1 and/or MnTBAP. (A) Cells treated with different concentrations of SIN-1. 24 hpi, virus was harvested and titers determined. (B) Cells treated with 400 μM SIN-1 and different concentrations of MnTBAP. Virus was harvested 24 hpi and titers determined. (C) Cells treated with different concentrations of MnTBAP, virus was harvested 24 hpi and titers determined.

    Article Snippet: Similarly, SARS-CoV infected Vero E6 cells were treated with a superoxide scavenger, Mn(III)tetrakis(4-Benzoic acid)porphyrin chloride (MnTBAP; Calbiochem), in the absence or presence of 400 μM SIN-1.

    Techniques: Infection

    Lysotracker Red-DND99 labels LAMP1 positive lysosomes in HeLa-mCC1a and Vero E6 cells. Scale bar 1μm.

    Journal: bioRxiv

    Article Title: β-Coronaviruses use lysosomal organelles for cellular egress

    doi: 10.1101/2020.07.25.192310

    Figure Lengend Snippet: Lysotracker Red-DND99 labels LAMP1 positive lysosomes in HeLa-mCC1a and Vero E6 cells. Scale bar 1μm.

    Article Snippet: Vero E6 cells were grown in Millicell EZ 8-well glass slides (Millipore) in infection media (EMEM, 4% FBS (Corning)) to a confluency of 90 – 100%.

    Techniques:

    Lysosomes are deacidified and lysosomal enzymes are inactive in β-Coronavirus infected cells. (A) Lysotracker Red DND99 staining of MHV-infected HeLa-mCC1a cells (12hr pi) and primary mouse macrophages (12hr pi); SARS-CoV2-infected Vero E6 cells (16hr pi). Representative images are presented. Scale 5μm for HeLa-CK and macrophage; 10μm for Vero E6 cell. (B) Mean Lysotracker Red fluorescence intensity per lysosome in MHV and CoV2 infected cells (n=20 cells for each condition; 10 lysosomes per cell). Bars are SE. (C) Number of Lysotracker Red positive organelles in mock or CoV2-infected Vero E6 cells (n=30 cells) were quantified and plotted. Bars are SE. (D) Lysosensor Green DND-199 was used to quantify the pH of lysosomes in MHV-infected HeLa-mCC1a cells. Mean fluorescence intensity of 10 lysosomes per mock (n=18) or MHV-infected (n=18) cell was converted to a pH value from calibration of dye. Bars are SD. (E) Lysosome enzyme activity measured in situ in mock- and MHV-infected HeLa-mCC1a cells. Cells were co-incubated with fluorogenic lysosomal enzyme substrate and 10kD dextran-coupled Alexa 555 dye. Plot presents mean fluorescence intensity of the enzyme substrate from similar mean dextran fluorescence intensity lysosomes (n=55 cells each). Bars are SD.

    Journal: bioRxiv

    Article Title: β-Coronaviruses use lysosomal organelles for cellular egress

    doi: 10.1101/2020.07.25.192310

    Figure Lengend Snippet: Lysosomes are deacidified and lysosomal enzymes are inactive in β-Coronavirus infected cells. (A) Lysotracker Red DND99 staining of MHV-infected HeLa-mCC1a cells (12hr pi) and primary mouse macrophages (12hr pi); SARS-CoV2-infected Vero E6 cells (16hr pi). Representative images are presented. Scale 5μm for HeLa-CK and macrophage; 10μm for Vero E6 cell. (B) Mean Lysotracker Red fluorescence intensity per lysosome in MHV and CoV2 infected cells (n=20 cells for each condition; 10 lysosomes per cell). Bars are SE. (C) Number of Lysotracker Red positive organelles in mock or CoV2-infected Vero E6 cells (n=30 cells) were quantified and plotted. Bars are SE. (D) Lysosensor Green DND-199 was used to quantify the pH of lysosomes in MHV-infected HeLa-mCC1a cells. Mean fluorescence intensity of 10 lysosomes per mock (n=18) or MHV-infected (n=18) cell was converted to a pH value from calibration of dye. Bars are SD. (E) Lysosome enzyme activity measured in situ in mock- and MHV-infected HeLa-mCC1a cells. Cells were co-incubated with fluorogenic lysosomal enzyme substrate and 10kD dextran-coupled Alexa 555 dye. Plot presents mean fluorescence intensity of the enzyme substrate from similar mean dextran fluorescence intensity lysosomes (n=55 cells each). Bars are SD.

    Article Snippet: Vero E6 cells were grown in Millicell EZ 8-well glass slides (Millipore) in infection media (EMEM, 4% FBS (Corning)) to a confluency of 90 – 100%.

    Techniques: Infection, Staining, Fluorescence, Activity Assay, In Situ, Incubation

    β-Coronaviruses exit cells independently of the biosynthetic secretory pathway. (A) Kinetics of MHV replication and release from HeLa-mCC1a cells. Viral genomic RNA quantified in cell lysates and extracellular medium with QPCR and plotted as fold increase over mock-infection. Experiments done in triplicates. Error bars are SD. (B) Impact of Brefeldin A (5μgr/ml) treatment between 8hr and 14hr pi on MHV release (black bar) or Gaussia Luciferase release (pink bar). Extracellular viral genomic RNA was quantified in extracellular medium with QPCR and plotted as fold increase over mock-infection. Experiments done in triplicates. Error bars are SD. (C) HeLa-mCC1a cells infected with MHV, fixed at 0,6,9,12hr pi and coimmunostained with anti-E (green) and anti-MHV (J1.3) (red) antibodies. Scale bar 5μm. (D) HeLa-mCC1a cells infected with MHV, fixed at 6,9,12hr pi and coimmunostained with anti-LAMP1 (green) and anti-MHV (J1.3) (red) antibodies. Arrows point to lysosomes containing M. Scale bar 5μm. (E) Immunoelectron micrographs of Rab7 positive lysosomes (black arrows) containing MHV particles (white arrows). After 12hr MHV infection, HeLa-mCC1a cells expressing Rab7-GFP were co-stained with anti-MHV (J1.3) and anti-GFP primary antibodies followed by 5nm and 10nm-gold coupled secondary antibodies respectively. Virus particles outlined in bottom panel. Scale bars indicated on micrographs. (F) Vero E6 cells infected with SARS-CoV2, fixed at 16hr pi and coimmunostained with anti-LAMP1 (green) and anti-CoV2 M (red) antibodies. Arrows point to lysosomes containing M. Scale bar 2μm. (G) Frequency of LAMP1 positive organelles in MHV-infected HeLa-mCC1a cells (n=16) at 12hr pi. In each cell, lysosomes within a 100μm 2 region of interest were scored for presence or absence of MHV and plotted. Error bars are SE. (H) Frequency of LAMP1 positive organelles in MHV-infected primary mouse macrophages (n=10) at 12hr pi. In each cell, lysosomes within a 100μm 2 region of interest were scored for presence or absence of MHV and plotted. Error bars are SE.

    Journal: bioRxiv

    Article Title: β-Coronaviruses use lysosomal organelles for cellular egress

    doi: 10.1101/2020.07.25.192310

    Figure Lengend Snippet: β-Coronaviruses exit cells independently of the biosynthetic secretory pathway. (A) Kinetics of MHV replication and release from HeLa-mCC1a cells. Viral genomic RNA quantified in cell lysates and extracellular medium with QPCR and plotted as fold increase over mock-infection. Experiments done in triplicates. Error bars are SD. (B) Impact of Brefeldin A (5μgr/ml) treatment between 8hr and 14hr pi on MHV release (black bar) or Gaussia Luciferase release (pink bar). Extracellular viral genomic RNA was quantified in extracellular medium with QPCR and plotted as fold increase over mock-infection. Experiments done in triplicates. Error bars are SD. (C) HeLa-mCC1a cells infected with MHV, fixed at 0,6,9,12hr pi and coimmunostained with anti-E (green) and anti-MHV (J1.3) (red) antibodies. Scale bar 5μm. (D) HeLa-mCC1a cells infected with MHV, fixed at 6,9,12hr pi and coimmunostained with anti-LAMP1 (green) and anti-MHV (J1.3) (red) antibodies. Arrows point to lysosomes containing M. Scale bar 5μm. (E) Immunoelectron micrographs of Rab7 positive lysosomes (black arrows) containing MHV particles (white arrows). After 12hr MHV infection, HeLa-mCC1a cells expressing Rab7-GFP were co-stained with anti-MHV (J1.3) and anti-GFP primary antibodies followed by 5nm and 10nm-gold coupled secondary antibodies respectively. Virus particles outlined in bottom panel. Scale bars indicated on micrographs. (F) Vero E6 cells infected with SARS-CoV2, fixed at 16hr pi and coimmunostained with anti-LAMP1 (green) and anti-CoV2 M (red) antibodies. Arrows point to lysosomes containing M. Scale bar 2μm. (G) Frequency of LAMP1 positive organelles in MHV-infected HeLa-mCC1a cells (n=16) at 12hr pi. In each cell, lysosomes within a 100μm 2 region of interest were scored for presence or absence of MHV and plotted. Error bars are SE. (H) Frequency of LAMP1 positive organelles in MHV-infected primary mouse macrophages (n=10) at 12hr pi. In each cell, lysosomes within a 100μm 2 region of interest were scored for presence or absence of MHV and plotted. Error bars are SE.

    Article Snippet: Vero E6 cells were grown in Millicell EZ 8-well glass slides (Millipore) in infection media (EMEM, 4% FBS (Corning)) to a confluency of 90 – 100%.

    Techniques: Real-time Polymerase Chain Reaction, Infection, Luciferase, Expressing, Staining