vero e6 cat crl 1586 cells  (ATCC)


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    ATCC vero e6 cat crl 1586 cells
    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. <t>Vero</t> <t>E6</t> cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).
    Vero E6 Cat Crl 1586 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vero e6 cat crl 1586 cells/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vero e6 cat crl 1586 cells - by Bioz Stars, 2024-05
    99/100 stars

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    1) Product Images from "Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro"

    Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30546-7

    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).
    Figure Legend Snippet: a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).

    Techniques Used: Activity Assay, Inhibition, Expressing, Quantitative RT-PCR, Infection, Sequencing

    a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .
    Figure Legend Snippet: a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Infection

    a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data  , calculated by two-tailed Student’s t test.
    Figure Legend Snippet: a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

    Techniques Used: Infection, Transfection, Quantitative RT-PCR, Staining, Two Tailed Test

    vero e6 cat crl 1586 cells  (ATCC)


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    ATCC vero e6 cat crl 1586 cells
    Vero E6 Cat Crl 1586 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86/100 stars

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    vero e6 cat crl 1586 cells  (ATCC)


    Bioz Verified Symbol ATCC is a verified supplier
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    Structured Review

    ATCC vero e6 cat crl 1586 cells
    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. <t>Vero</t> <t>E6</t> cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).
    Vero E6 Cat Crl 1586 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vero e6 cat crl 1586 cells/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vero e6 cat crl 1586 cells - by Bioz Stars, 2024-05
    99/100 stars

    Images

    1) Product Images from "Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro"

    Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30546-7

    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).
    Figure Legend Snippet: a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).

    Techniques Used: Activity Assay, Inhibition, Expressing, Quantitative RT-PCR, Infection, Sequencing

    a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .
    Figure Legend Snippet: a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Infection

    a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data  , calculated by two-tailed Student’s t test.
    Figure Legend Snippet: a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

    Techniques Used: Infection, Transfection, Quantitative RT-PCR, Staining, Two Tailed Test

    vero e6 cat crl 1586 cell lines  (ATCC)


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    ATCC vero e6 cat crl 1586 cell lines
    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on <t>Vero</t> <t>E6</t> cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant
    Vero E6 Cat Crl 1586 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vero e6 cat crl 1586 cell lines/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vero e6 cat crl 1586 cell lines - by Bioz Stars, 2024-05
    99/100 stars

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    1) Product Images from "ACE2‐enriched extracellular vesicles enhance infectivity of live SARS‐CoV‐2 virus"

    Article Title: ACE2‐enriched extracellular vesicles enhance infectivity of live SARS‐CoV‐2 virus

    Journal: Journal of Extracellular Vesicles

    doi: 10.1002/jev2.12231

    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on Vero E6 cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant
    Figure Legend Snippet: ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on Vero E6 cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant

    Techniques Used: Infection, Western Blot, Software, Purification, Growth Assay, Quantitative RT-PCR

    Inhibition of EV uptake reduces infectivity of SARS‐CoV‐2 on Vero E6 cells. (a) Vero E6 cells were pretreated with inhibitors including EIPA, Cytochalasin, Filipin and BafA1 followed by incubation with PKH67‐labeled EVs of 293T ACE2 overexpressing cells. Cells were fixed and stained with DAPI. Representative fluorescent images captured by confocal microscope are shown. Scale bar: 20 μm. (b) The fluorescent signal was analysed using ZEN Software Version 3.1. Error bars represent mean ± SEM. *** P < 0.001. (c) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated EVs (1:8 dilution) and cytochalasin D and BafA1. The infectious media were collected to determine viral load by RT‐qPCR. Representative data with error bar represents mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. * P < 0.05, *** P < 0.001, **** P < 0.0001. (d) Schematic illustration of the proposed cell entry mechanism of SARS‐CoV‐2 mediated by EV‐ACE2. (a) SARS‐CoV‐2 spike (S) protein binds ACE2 on host cells followed by activation of S protein by host protease TMPRSS2. Cytochalasin D blocks infection of host cells by SARS‐CoA‐2. (b) Proteolytic cleavage of ACE2 releases secretory ACE2 (sACE2) which facilitates virus cell entry through AVPR1B and/or AT1 receptor. (c) ACE2 on the surface of EV binds S protein and assists cell entry of virus through EV uptake mechanism that can be inhibited by cytochalasin D (inhibitor of actin polymerization) and BafA1 (inhibitor of vacuolar H+ ATPase). (d) ACE2 carried by EV acts as a decoy to bind virus, thus limits the cell entry of virus
    Figure Legend Snippet: Inhibition of EV uptake reduces infectivity of SARS‐CoV‐2 on Vero E6 cells. (a) Vero E6 cells were pretreated with inhibitors including EIPA, Cytochalasin, Filipin and BafA1 followed by incubation with PKH67‐labeled EVs of 293T ACE2 overexpressing cells. Cells were fixed and stained with DAPI. Representative fluorescent images captured by confocal microscope are shown. Scale bar: 20 μm. (b) The fluorescent signal was analysed using ZEN Software Version 3.1. Error bars represent mean ± SEM. *** P < 0.001. (c) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated EVs (1:8 dilution) and cytochalasin D and BafA1. The infectious media were collected to determine viral load by RT‐qPCR. Representative data with error bar represents mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. * P < 0.05, *** P < 0.001, **** P < 0.0001. (d) Schematic illustration of the proposed cell entry mechanism of SARS‐CoV‐2 mediated by EV‐ACE2. (a) SARS‐CoV‐2 spike (S) protein binds ACE2 on host cells followed by activation of S protein by host protease TMPRSS2. Cytochalasin D blocks infection of host cells by SARS‐CoA‐2. (b) Proteolytic cleavage of ACE2 releases secretory ACE2 (sACE2) which facilitates virus cell entry through AVPR1B and/or AT1 receptor. (c) ACE2 on the surface of EV binds S protein and assists cell entry of virus through EV uptake mechanism that can be inhibited by cytochalasin D (inhibitor of actin polymerization) and BafA1 (inhibitor of vacuolar H+ ATPase). (d) ACE2 carried by EV acts as a decoy to bind virus, thus limits the cell entry of virus

    Techniques Used: Inhibition, Infection, Incubation, Labeling, Staining, Microscopy, Software, Quantitative RT-PCR, Activation Assay

    cell lines cell line source s vero e6 cell line atcc cat crl 1586  (ATCC)


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    ATCC vero e6 cat crl 1586 cells
    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. <t>Vero</t> <t>E6</t> cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).
    Vero E6 Cat Crl 1586 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC vero e6 cat crl 1586 cell lines
    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on <t>Vero</t> <t>E6</t> cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant
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    ATCC cell lines cell line source s vero e6 cell line atcc cat crl 1586
    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on <t>Vero</t> <t>E6</t> cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant
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    ATCC cell lines african green monkey vero e6 cells atcc cat
    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on <t>Vero</t> <t>E6</t> cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant
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    a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).

    Journal: Nature Communications

    Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

    doi: 10.1038/s41467-022-30546-7

    Figure Lengend Snippet: a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).

    Article Snippet: Human embryonic kidney (HEK293T, Cat# CRL-3216), A549 (Cat# CCL-185), MRC-5 (Cat# CCL-171), and Vero E6 (Cat# CRL-1586) cells were purchased from ATCC and cultured in 10% fetal bovine serum (Thermo Fisher Scientific) in DMEM (Thermo Fisher Scientific).

    Techniques: Activity Assay, Inhibition, Expressing, Quantitative RT-PCR, Infection, Sequencing

    a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .

    Journal: Nature Communications

    Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

    doi: 10.1038/s41467-022-30546-7

    Figure Lengend Snippet: a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .

    Article Snippet: Human embryonic kidney (HEK293T, Cat# CRL-3216), A549 (Cat# CCL-185), MRC-5 (Cat# CCL-171), and Vero E6 (Cat# CRL-1586) cells were purchased from ATCC and cultured in 10% fetal bovine serum (Thermo Fisher Scientific) in DMEM (Thermo Fisher Scientific).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Infection

    a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data  , calculated by two-tailed Student’s t test.

    Journal: Nature Communications

    Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

    doi: 10.1038/s41467-022-30546-7

    Figure Lengend Snippet: a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

    Article Snippet: Human embryonic kidney (HEK293T, Cat# CRL-3216), A549 (Cat# CCL-185), MRC-5 (Cat# CCL-171), and Vero E6 (Cat# CRL-1586) cells were purchased from ATCC and cultured in 10% fetal bovine serum (Thermo Fisher Scientific) in DMEM (Thermo Fisher Scientific).

    Techniques: Infection, Transfection, Quantitative RT-PCR, Staining, Two Tailed Test

    ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on Vero E6 cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant

    Journal: Journal of Extracellular Vesicles

    Article Title: ACE2‐enriched extracellular vesicles enhance infectivity of live SARS‐CoV‐2 virus

    doi: 10.1002/jev2.12231

    Figure Lengend Snippet: ACE2‐enriched EVs increase infectivity of live SARS‐CoV‐2 on Vero E6 cells. (a) Western blot analysis of ACE2 in the total cell lysate (TCL), extracellular vesicle (EV) and conditioned medium (CM) of control (CTL) and stable ACE2 overexpressing (ACE2‐OE) cells. Positive (TSG101) and negative (GM130 and α‐tubulin) markers of small EVs were examined. Arrowheads indicate the expressions of different forms of ACE2. (b) Expressions of TMPRSS2 ( left ) and TMPRSS4 ( right ) in control and ACE2 overexpressing cells. Arrowheads indicate the expressions of different forms of TMPRSS2 and TMPRSS4. (c) Size range of EVs was measured by nanoparticle tracking analyser. Automated measurements taken at 11 distinct positions with standard instrument setting (sensitivity:75, shutter: 100, min. brightness: 30; min. area: 10; max area: 1000; fps: 30) in the sample cell and the peak analysis was performed by ZetaView software. The mode size is displayed by font in red. (d) EVs were purified from medium of control and ACE2 overexpressing cells. EVs were subjected to immunogold labelling with or without anti‐ACE2 antibody with secondary antibodies conjugated to 15‐nm gold particles. Representative electron micrographs of EVs. Scale bar: 100 nm. (e) Schematic diagram illustrating the multicycle growth assay. (f) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated diluted EVs. The infectious media were collected to determine viral load by RT‐qPCR. The data represent the mean of 2 independent experiments with 3 biological samples each. Error bars represent mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. ** P < 0.01, **** P < 0.0001. NS , Not significant

    Article Snippet: 293T (Cat# CRL‐3216) and Vero E6 (Cat# CRL‐1586) cell lines were purchased from American Type Culture Collection.

    Techniques: Infection, Western Blot, Software, Purification, Growth Assay, Quantitative RT-PCR

    Inhibition of EV uptake reduces infectivity of SARS‐CoV‐2 on Vero E6 cells. (a) Vero E6 cells were pretreated with inhibitors including EIPA, Cytochalasin, Filipin and BafA1 followed by incubation with PKH67‐labeled EVs of 293T ACE2 overexpressing cells. Cells were fixed and stained with DAPI. Representative fluorescent images captured by confocal microscope are shown. Scale bar: 20 μm. (b) The fluorescent signal was analysed using ZEN Software Version 3.1. Error bars represent mean ± SEM. *** P < 0.001. (c) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated EVs (1:8 dilution) and cytochalasin D and BafA1. The infectious media were collected to determine viral load by RT‐qPCR. Representative data with error bar represents mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. * P < 0.05, *** P < 0.001, **** P < 0.0001. (d) Schematic illustration of the proposed cell entry mechanism of SARS‐CoV‐2 mediated by EV‐ACE2. (a) SARS‐CoV‐2 spike (S) protein binds ACE2 on host cells followed by activation of S protein by host protease TMPRSS2. Cytochalasin D blocks infection of host cells by SARS‐CoA‐2. (b) Proteolytic cleavage of ACE2 releases secretory ACE2 (sACE2) which facilitates virus cell entry through AVPR1B and/or AT1 receptor. (c) ACE2 on the surface of EV binds S protein and assists cell entry of virus through EV uptake mechanism that can be inhibited by cytochalasin D (inhibitor of actin polymerization) and BafA1 (inhibitor of vacuolar H+ ATPase). (d) ACE2 carried by EV acts as a decoy to bind virus, thus limits the cell entry of virus

    Journal: Journal of Extracellular Vesicles

    Article Title: ACE2‐enriched extracellular vesicles enhance infectivity of live SARS‐CoV‐2 virus

    doi: 10.1002/jev2.12231

    Figure Lengend Snippet: Inhibition of EV uptake reduces infectivity of SARS‐CoV‐2 on Vero E6 cells. (a) Vero E6 cells were pretreated with inhibitors including EIPA, Cytochalasin, Filipin and BafA1 followed by incubation with PKH67‐labeled EVs of 293T ACE2 overexpressing cells. Cells were fixed and stained with DAPI. Representative fluorescent images captured by confocal microscope are shown. Scale bar: 20 μm. (b) The fluorescent signal was analysed using ZEN Software Version 3.1. Error bars represent mean ± SEM. *** P < 0.001. (c) Vero E6 cells were infected with SARS‐CoV‐2 premixed with the indicated EVs (1:8 dilution) and cytochalasin D and BafA1. The infectious media were collected to determine viral load by RT‐qPCR. Representative data with error bar represents mean ± SEM. Asterisks indicate statistical significance compared with PBS control group. * P < 0.05, *** P < 0.001, **** P < 0.0001. (d) Schematic illustration of the proposed cell entry mechanism of SARS‐CoV‐2 mediated by EV‐ACE2. (a) SARS‐CoV‐2 spike (S) protein binds ACE2 on host cells followed by activation of S protein by host protease TMPRSS2. Cytochalasin D blocks infection of host cells by SARS‐CoA‐2. (b) Proteolytic cleavage of ACE2 releases secretory ACE2 (sACE2) which facilitates virus cell entry through AVPR1B and/or AT1 receptor. (c) ACE2 on the surface of EV binds S protein and assists cell entry of virus through EV uptake mechanism that can be inhibited by cytochalasin D (inhibitor of actin polymerization) and BafA1 (inhibitor of vacuolar H+ ATPase). (d) ACE2 carried by EV acts as a decoy to bind virus, thus limits the cell entry of virus

    Article Snippet: 293T (Cat# CRL‐3216) and Vero E6 (Cat# CRL‐1586) cell lines were purchased from American Type Culture Collection.

    Techniques: Inhibition, Infection, Incubation, Labeling, Staining, Microscopy, Software, Quantitative RT-PCR, Activation Assay