sars cov 2 2019 ncov nucleocapsid antibody rabbit pab  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Nucleocapsid Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Nucleocapsid Protein Catalog 40588 V08B YP 009724397 2 335Gly Ala Met1 Ala419 The specific IgG was purified by SARS CoV 2 2019 nCoV Nucleocapsid affinity chromatography
    Catalog Number:
    40588-T62
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    2019 nCoV
    Applications:
    WB,ELISA
    Immunogen:
    Recombinant SARS-CoV-2 / 2019-nCoV Nucleocapsid Protein (Catalog#40588-V08B)
    Product Aliases:
    Anti-coronavirus NP Antibody, Anti-coronavirus Nucleocapsid Antibody, Anti-coronavirus Nucleoprotein Antibody, Anti-cov np Antibody, Anti-ncov NP Antibody, Anti-NCP-CoV Nucleocapsid Antibody, Anti-novel coronavirus NP Antibody, Anti-novel coronavirus Nucleocapsid Antibody, Anti-novel coronavirus Nucleoprotein Antibody, Anti-np Antibody, Anti-nucleocapsid Antibody, Anti-Nucleoprotein Antibody
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological sars cov 2 2019 ncov nucleocapsid antibody rabbit pab
    A wide range of ACE2 orthologs support binding to RBD proteins of <t>SARS-CoV-2</t> and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Nucleocapsid Protein Catalog 40588 V08B YP 009724397 2 335Gly Ala Met1 Ala419 The specific IgG was purified by SARS CoV 2 2019 nCoV Nucleocapsid affinity chromatography
    https://www.bioz.com/result/sars cov 2 2019 ncov nucleocapsid antibody rabbit pab/product/Sino Biological
    Average 99 stars, based on 1 article reviews
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    sars cov 2 2019 ncov nucleocapsid antibody rabbit pab - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig"

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    Journal: Journal of Virology

    doi: 10.1128/JVI.01283-20

    A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.
    Figure Legend Snippet: A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

    Techniques Used: Binding Assay, Transfection, Expressing, Staining, Flow Cytometry, Western Blot

    A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.
    Figure Legend Snippet: A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

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

    The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.
    Figure Legend Snippet: The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

    Techniques Used: Variant Assay, Expressing, Infection, Luciferase, Mutagenesis, Neutralization, Activity Assay

    Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P
    Figure Legend Snippet: Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

    Techniques Used: Recombinant, Blocking Assay, Expressing, Infection, Purification, Luciferase, Concentration Assay, Two Tailed Test

    SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues
    Figure Legend Snippet: SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

    Techniques Used: Binding Assay

    A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.
    Figure Legend Snippet: A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

    Techniques Used: Variant Assay, Expressing, Infection, Luciferase, Derivative Assay, Staining, Fluorescence, Microscopy

    2) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Figure Legend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P
    Figure Legend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Negative Control

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    3) Product Images from "Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection"

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2021.03.03.433675

    Transcriptional response to SARS-CoV-2 infection of Calu3 cells is dominated by antiviral defense genes (A) Volcano plot showing global transcriptional changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value
    Figure Legend Snippet: Transcriptional response to SARS-CoV-2 infection of Calu3 cells is dominated by antiviral defense genes (A) Volcano plot showing global transcriptional changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Techniques Used: Infection

    MNAse digestion of cell extracts yields 30nt long CDS-mapped ribosome footprints. (A) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 hours. (B) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. (C-D) Metagene coverage aligned to the start codon of all annotated protein coding genes for mock and SARS-CoV-2 infected cells. (E-F) Read-length counts per million read distribution in coding sequence and 5’ untranslated regions (UTR).
    Figure Legend Snippet: MNAse digestion of cell extracts yields 30nt long CDS-mapped ribosome footprints. (A) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 hours. (B) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. (C-D) Metagene coverage aligned to the start codon of all annotated protein coding genes for mock and SARS-CoV-2 infected cells. (E-F) Read-length counts per million read distribution in coding sequence and 5’ untranslated regions (UTR).

    Techniques Used: Infection, Sequencing

    Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. (A) Volcano plot showing global translational changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value
    Figure Legend Snippet: Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. (A) Volcano plot showing global translational changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Techniques Used: Infection

    SARS-CoV-2 infection of Calu-3 cells (A) TCID 50 measurements of virus titres, (B) qRT-PCR measurements of intracellular viral RNA represented by 2 -ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and (C) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). (D) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infection of Calu-3 cells (A) TCID 50 measurements of virus titres, (B) qRT-PCR measurements of intracellular viral RNA represented by 2 -ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and (C) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). (D) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).

    Techniques Used: Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining

    Unstable genes are more sensitive to translation inhibition. (A) Volcano plot showing global translation efficiency (Riborex engine) changes of 6,878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 hours versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value
    Figure Legend Snippet: Unstable genes are more sensitive to translation inhibition. (A) Volcano plot showing global translation efficiency (Riborex engine) changes of 6,878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 hours versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Techniques Used: Inhibition, Infection

    4) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    5) Product Images from "Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19"

    Article Title: Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.04.008

    Suppressive effects of dipyridamole (DIP) and chloroquine on SARS-CoV-2 replication in vitro . (A) Chemical structure of DIP. (B) Enzyme activity of Mpro in the presence of ascending concentrations of DIP. (C) Dose-dependent suppression of SARS-CoV-2 replication by DIP and chloroquine in vitro . Virus titers were measured by Foci forming assay, inhibition rates were performed by indirect immunoinfluscent assay, and calculated inhibition rates of different dosages of DIP or chloroquine were compared with virus control. P values were calculated by ANOVA.
    Figure Legend Snippet: Suppressive effects of dipyridamole (DIP) and chloroquine on SARS-CoV-2 replication in vitro . (A) Chemical structure of DIP. (B) Enzyme activity of Mpro in the presence of ascending concentrations of DIP. (C) Dose-dependent suppression of SARS-CoV-2 replication by DIP and chloroquine in vitro . Virus titers were measured by Foci forming assay, inhibition rates were performed by indirect immunoinfluscent assay, and calculated inhibition rates of different dosages of DIP or chloroquine were compared with virus control. P values were calculated by ANOVA.

    Techniques Used: In Vitro, Activity Assay, Inhibition

    6) Product Images from "Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies"

    Article Title: Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies

    Journal: Cell Research

    doi: 10.1038/s41422-021-00487-9

    The classification and epitope of the 10 anti-SARS-CoV-2 nAbs. The 10 antibodies could be classified into three groups (G1, group 1; G2, group 2; G3, group 3). Group 1 antibodies can be further divided into three subgroups (sub1, sub2, sub3). The complexes of RBD with ACE2 or nAbs are shown as cartoon with RBD colored in wheat, the light chains of nAbs colored in cyan and the heavy chains of nAbs colored in different colors. For the epitope display, RBD is shown as gray surface in top, front and back views, with interface that binds to ACE2 colored in cyan and the epitopes of different nAbs shown in respective colors. For the top views, the epitopes corresponding to heavy chains are shown in respective colors (Top view Hc) and epitopes corresponding to light chains are shown in cyan (Top view Lc). Hc, heavy chain; Lc, light chain. The “Overlapped epitopes” column displays the residue number of nAb epitope that overlaps with ACE2-binding site. The “Heavy chain” and “Light chain” columns show the residue number of the epitope of the respective chain that overlaps with ACE2-binding site. The binding affinity to RBD of IgG antibodies was also shown.
    Figure Legend Snippet: The classification and epitope of the 10 anti-SARS-CoV-2 nAbs. The 10 antibodies could be classified into three groups (G1, group 1; G2, group 2; G3, group 3). Group 1 antibodies can be further divided into three subgroups (sub1, sub2, sub3). The complexes of RBD with ACE2 or nAbs are shown as cartoon with RBD colored in wheat, the light chains of nAbs colored in cyan and the heavy chains of nAbs colored in different colors. For the epitope display, RBD is shown as gray surface in top, front and back views, with interface that binds to ACE2 colored in cyan and the epitopes of different nAbs shown in respective colors. For the top views, the epitopes corresponding to heavy chains are shown in respective colors (Top view Hc) and epitopes corresponding to light chains are shown in cyan (Top view Lc). Hc, heavy chain; Lc, light chain. The “Overlapped epitopes” column displays the residue number of nAb epitope that overlaps with ACE2-binding site. The “Heavy chain” and “Light chain” columns show the residue number of the epitope of the respective chain that overlaps with ACE2-binding site. The binding affinity to RBD of IgG antibodies was also shown.

    Techniques Used: Binding Assay

    Neutralizing activity and shedding of S1 by IgG- and Fab-forms of nAbs. a Neutralizing activity against SARS-CoV-2 pseudovirus by P5A-1B8, P5A-1B9, and P5A-2G7 in IgG-forms (solid line) and Fab-forms (dotted line). Data were representative of at least two independent experiments. b Shedding of S1 over time measured using flow cytometry at 37 °C with 293T cell-surface expressed wild-type SARS-Cov-2 S protein. c Similar to b , with a mutant S protein containing GSAS substitution at S1/S2 cleavage site. Data were from three independent experiments, shown as means ± SEM.
    Figure Legend Snippet: Neutralizing activity and shedding of S1 by IgG- and Fab-forms of nAbs. a Neutralizing activity against SARS-CoV-2 pseudovirus by P5A-1B8, P5A-1B9, and P5A-2G7 in IgG-forms (solid line) and Fab-forms (dotted line). Data were representative of at least two independent experiments. b Shedding of S1 over time measured using flow cytometry at 37 °C with 293T cell-surface expressed wild-type SARS-Cov-2 S protein. c Similar to b , with a mutant S protein containing GSAS substitution at S1/S2 cleavage site. Data were from three independent experiments, shown as means ± SEM.

    Techniques Used: Activity Assay, Flow Cytometry, Mutagenesis

    7) Product Images from "Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell"

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

    Journal: bioRxiv

    doi: 10.1101/2021.03.01.433431

    The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p
    Figure Legend Snippet: The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p
    Figure Legend Snippet: MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p

    Techniques Used: Infection, Quantitative RT-PCR, Transfection

    H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value
    Figure Legend Snippet: H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value

    Techniques Used: Infection, Expressing

    The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p
    Figure Legend Snippet: The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p

    Techniques Used: Infection, Expressing, Blocking Assay, Quantitative RT-PCR

    H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.
    Figure Legend Snippet: H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.

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

    The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p
    Figure Legend Snippet: The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p

    Techniques Used: Expressing, Infection, RNA Sequencing Assay, Quantitative RT-PCR, Software, Western Blot, Titration

    H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.
    Figure Legend Snippet: H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.

    Techniques Used: Infection, Labeling, Liquid Chromatography, Mass Spectrometry

    8) Product Images from "ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2"

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.11.13.381343

    ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p
    Figure Legend Snippet: ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Techniques Used: Infection, Transfection

    Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p
    Figure Legend Snippet: Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Techniques Used: Infection, Transfection

    9) Product Images from "Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell"

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

    Journal: bioRxiv

    doi: 10.1101/2021.03.01.433431

    The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p
    Figure Legend Snippet: The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p
    Figure Legend Snippet: MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p

    Techniques Used: Infection, Quantitative RT-PCR, Transfection

    H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value
    Figure Legend Snippet: H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value

    Techniques Used: Infection, Expressing

    The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p
    Figure Legend Snippet: The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p

    Techniques Used: Infection, Expressing, Blocking Assay, Quantitative RT-PCR

    H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.
    Figure Legend Snippet: H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.

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

    The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p
    Figure Legend Snippet: The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p

    Techniques Used: Expressing, Infection, RNA Sequencing Assay, Quantitative RT-PCR, Software, Western Blot, Titration

    H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.
    Figure Legend Snippet: H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.

    Techniques Used: Infection, Labeling, Liquid Chromatography, Mass Spectrometry

    10) Product Images from "Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery"

    Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

    Journal: bioRxiv

    doi: 10.1101/2020.08.02.233064

    Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.
    Figure Legend Snippet: Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.

    Techniques Used: Library Screening, Infection, Plasmid Preparation, Generated, Electric Cell-substrate Impedance Sensing, Concentration Assay, Staining

    SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors
    Figure Legend Snippet: SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors

    Techniques Used: Infection, Modification, Immunostaining, Plasmid Preparation, Generated

    11) Product Images from "Ribosome-Profiling Reveals Restricted Post Transcriptional Expression of Antiviral Cytokines and Transcription Factors during SARS-CoV-2 Infection"

    Article Title: Ribosome-Profiling Reveals Restricted Post Transcriptional Expression of Antiviral Cytokines and Transcription Factors during SARS-CoV-2 Infection

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22073392

    mRNA features influence sensitivity to translation inhibition. ( A ) Volcano plot showing global translation efficiency (Riborex engine) changes of 6878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 h versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value
    Figure Legend Snippet: mRNA features influence sensitivity to translation inhibition. ( A ) Volcano plot showing global translation efficiency (Riborex engine) changes of 6878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 h versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value

    Techniques Used: Inhibition, Infection

    SARS-CoV-2 infection of Calu-3 cells ( A ) TCID50 measurements of virus titres, ( B ) qRT-PCR measurements of intracellular viral RNA represented by 2-ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and ( C ) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). ( D ) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infection of Calu-3 cells ( A ) TCID50 measurements of virus titres, ( B ) qRT-PCR measurements of intracellular viral RNA represented by 2-ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and ( C ) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). ( D ) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).

    Techniques Used: Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining

    MNAse digestion of cell extracts yields 30 nt long CDS-mapped ribosome footprints. ( A ) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 h. ( B ) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. ( C , D ) Read-length counts per million read distribution in coding sequence and 5′ untranslated regions (UTR).
    Figure Legend Snippet: MNAse digestion of cell extracts yields 30 nt long CDS-mapped ribosome footprints. ( A ) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 h. ( B ) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. ( C , D ) Read-length counts per million read distribution in coding sequence and 5′ untranslated regions (UTR).

    Techniques Used: Infection, Sequencing

    Transcriptional response to SARS-CoV-2 infection of Calu-3 cells is dominated by antiviral defense genes. ( A ) Volcano plot showing global transcriptional changes of ~11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 h versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value
    Figure Legend Snippet: Transcriptional response to SARS-CoV-2 infection of Calu-3 cells is dominated by antiviral defense genes. ( A ) Volcano plot showing global transcriptional changes of ~11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 h versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value

    Techniques Used: Infection

    Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. ( A ) Volcano plot showing global translational changes of ~11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 h versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value
    Figure Legend Snippet: Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. ( A ) Volcano plot showing global translational changes of ~11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 h versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p -value

    Techniques Used: Infection

    12) Product Images from "Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell"

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

    Journal: bioRxiv

    doi: 10.1101/2021.03.01.433431

    The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p
    Figure Legend Snippet: The SARS-CoV-2 S protein is necessary but not sufficient for viral entry in the H522 cell line. A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p
    Figure Legend Snippet: MDA5 mediates the IFN response to SARS-CoV-2 infection. A , Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n=2). β-actin represents the loading control. B , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were either mock transfected or transfected with a non-targeting (NT) siRNA 24 h prior to infection. C , ISG mRNA levels was detected by qRT-PCR in H522 cells infected with SARS-CoV-2 96 hpi. H522 cells were transfected with a non-targeting (NT) siRNA or a panel of siRNAs targeting genes involved in viral sensing 24 h prior to infection. * indicates p

    Techniques Used: Infection, Quantitative RT-PCR, Transfection

    H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value
    Figure Legend Snippet: H522 transcriptome response to SARS-CoV-2 infection. A , Experimental design of transcriptomics experiments. H522 cells were infected with SARS-CoV-2 at MOI 1.0, 0.25, 0.06, or 0.015 and harvested after 4, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 h. All conditions were performed in duplicate. B , Relative expression of SARS-CoV-2 RNA vs. H. sapiens RNA from H522 (n=2). C , Principle component analysis of highly expressed genes from MOIs 0.25 and 1 across all time points. D , Volcano plot of gene expression changes comparing mock infection to 96 hours post infection of MOIs=0.25 and 1. Select changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S2. E , Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log 2 fold change > 2 and adjusted p-value

    Techniques Used: Infection, Expressing

    The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p
    Figure Legend Snippet: The H522 cell line is permissive to SARS-CoV-2 infection independent of ACE2 expression. A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p

    Techniques Used: Infection, Expressing, Blocking Assay, Quantitative RT-PCR

    H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.
    Figure Legend Snippet: H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins. A , H522 cells were pre-treated with increasing concentrations of bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at MOI=1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO treated cells (n≥3). See also Figure S4. B , Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 over time (representative of n=2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4 hours mock control. Quantification was performed using the Licor Image Studio software and values are indicated below the immunoblots. C , Basal HBECs from 5 different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in the presence of the inhibitor. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi and normalized to DMSO treated cells.

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

    The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p
    Figure Legend Snippet: The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection. A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL , and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p

    Techniques Used: Expressing, Infection, RNA Sequencing Assay, Quantitative RT-PCR, Software, Western Blot, Titration

    H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.
    Figure Legend Snippet: H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways. A , Experimental design of proteomics experiments. H522 cells were infected with SARS-CoV-2 at MOI=1 and harvested after 4, 12, 24, 48, 72, and 96 h. Mock-infected cells were harvested after 4 and 96 h. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. B , Principal component analysis of whole cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n=3). C, Quantification of total ion intensities for each identified SARS-CoV-2 protein over time and normalized to the 4 h mock control. The shaded grey regions represent SEM. D , Volcano plot of protein abundance at 96hpi compared to th e 96 h mock control. See also Table S4 E , Differentially expressed proteins from ‘D’ were clustered based on z-score. F , Quantification of total ion intensities normalized to the 4 h mock control for each protein across the 7 identified clusters in ‘D’. The colored lines represent quantification of an individual protein whereas the solid black and dashed black lines represent the mean of infected and mock samples, respectively. G , Hypergeometric enrichment analysis from three different databases for each individual cluster in ‘D’ (Hallmark, Reactome, Gene Ontology). The color of the circle represents significance (q-value), whereas the size of the circle indicates the percentage of the cluster represented in the pathway. See also Table S5. H , Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log 2 fold change over 4 h mock for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, 96 hpi. I , Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. J , Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5. K , Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID Multi-Validated Datasets. Interferon related functions were extracted from GO terms in MSigDB. The colors correspond to the whole cell proteomic clusters identified in ‘D’. See also Figure S5.

    Techniques Used: Infection, Labeling, Liquid Chromatography, Mass Spectrometry

    Related Articles

    Staining:

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell
    Article Snippet: For FACS, cells were detached then fixed with 4% PFA for 20 min at room temperature, followed by permeabilization using 0.5% Tween-20 in PBS for 10 min. .. Cells were blocked with 1% bovine serum albumin (BSA) and 10% FBS in 0.1% Tween-20 PBS (PBST) for 1 h prior to staining with a rabbit polyclonal anti SARS-CoV-2 nucleocapsid antibody (Sino Biological Inc. catalog # 40588-T62) diluted 1:500 and incubated overnight at 4°C. .. The following day, after washed cells were stained with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) at 1:1000 dilution.

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2
    Article Snippet: .. Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008). .. Nuclei were counter-stained with diamidino-2-phenylindole (DAPI).

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection
    Article Snippet: .. Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008). ..

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: 293T cells expressing one of the 16 ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 50% tissue culture infective dose(s) (TCID50) and incubated for 1 h at 37°C. .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Incubation:

    Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell
    Article Snippet: For FACS, cells were detached then fixed with 4% PFA for 20 min at room temperature, followed by permeabilization using 0.5% Tween-20 in PBS for 10 min. .. Cells were blocked with 1% bovine serum albumin (BSA) and 10% FBS in 0.1% Tween-20 PBS (PBST) for 1 h prior to staining with a rabbit polyclonal anti SARS-CoV-2 nucleocapsid antibody (Sino Biological Inc. catalog # 40588-T62) diluted 1:500 and incubated overnight at 4°C. .. The following day, after washed cells were stained with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) at 1:1000 dilution.

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: 293T cells expressing one of the 16 ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 50% tissue culture infective dose(s) (TCID50) and incubated for 1 h at 37°C. .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Article Title: Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies
    Article Snippet: The plates were then incubated at 37 °C for 24 h. Overlays were removed and then cells were fixed with 4% paraformaldehyde solution for 30 min, permeabilized with Perm/Wash buffer (BD Biosciences) containing 0.1% Triton X-100 for 10 min. .. Cells were incubated with rabbit anti-SARS-CoV-2 NP IgG (Sino Biological Inc.) for 1 h at room temperature before adding HRP-conjugated goat anti-rabbit IgG (H + L) antibody (TransGen Biotech, Beijing). .. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences Inc.).

    Article Title: Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19
    Article Snippet: After 24 h incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. .. And then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological, Inc., Beijing, China), followed by an HRP-labelled goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). .. The foci were visualized by TrueBlue™ Peroxidase Substrate (KPL, Gaithersburg, MD, USA), and counted with an ELISPOT reader (CTL, Shaker Heights, OH, USA).

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity
    Article Snippet: To reduce proteins, DTT (final concentration 62.5 mM) was added to the 4× Laemmli sample buffer (Bio-Rad). .. The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ). .. Secondary antibodies used were donkey anti-rabbit HRP (ab16284, Abcam, 1:2000) in 2% BSA TBS/T.

    Immunofluorescence:

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2
    Article Snippet: .. Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008). .. Nuclei were counter-stained with diamidino-2-phenylindole (DAPI).

    Fluorescence:

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: 293T cells expressing one of the 16 ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 50% tissue culture infective dose(s) (TCID50) and incubated for 1 h at 37°C. .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Microscopy:

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: 293T cells expressing one of the 16 ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 50% tissue culture infective dose(s) (TCID50) and incubated for 1 h at 37°C. .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

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    Sino Biological sars cov 2 2019 ncov nucleocapsid antibody rabbit pab
    A wide range of ACE2 orthologs support binding to RBD proteins of <t>SARS-CoV-2</t> and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.
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    A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Binding Assay, Transfection, Expressing, Staining, Flow Cytometry, Western Blot

    A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

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

    The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Variant Assay, Expressing, Infection, Luciferase, Mutagenesis, Neutralization, Activity Assay

    Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Recombinant, Blocking Assay, Expressing, Infection, Purification, Luciferase, Concentration Assay, Two Tailed Test

    SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Binding Assay

    A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Variant Assay, Expressing, Infection, Luciferase, Derivative Assay, Staining, Fluorescence, Microscopy

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Journal: JCI Insight

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    doi: 10.1172/jci.insight.142386

    Figure Lengend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Journal: JCI Insight

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    doi: 10.1172/jci.insight.142386

    Figure Lengend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Binding Assay, Polymerase Chain Reaction, Negative Control

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Journal: JCI Insight

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    doi: 10.1172/jci.insight.142386

    Figure Lengend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    Transcriptional response to SARS-CoV-2 infection of Calu3 cells is dominated by antiviral defense genes (A) Volcano plot showing global transcriptional changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Journal: bioRxiv

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    doi: 10.1101/2021.03.03.433675

    Figure Lengend Snippet: Transcriptional response to SARS-CoV-2 infection of Calu3 cells is dominated by antiviral defense genes (A) Volcano plot showing global transcriptional changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection

    MNAse digestion of cell extracts yields 30nt long CDS-mapped ribosome footprints. (A) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 hours. (B) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. (C-D) Metagene coverage aligned to the start codon of all annotated protein coding genes for mock and SARS-CoV-2 infected cells. (E-F) Read-length counts per million read distribution in coding sequence and 5’ untranslated regions (UTR).

    Journal: bioRxiv

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    doi: 10.1101/2021.03.03.433675

    Figure Lengend Snippet: MNAse digestion of cell extracts yields 30nt long CDS-mapped ribosome footprints. (A) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 hours. (B) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. (C-D) Metagene coverage aligned to the start codon of all annotated protein coding genes for mock and SARS-CoV-2 infected cells. (E-F) Read-length counts per million read distribution in coding sequence and 5’ untranslated regions (UTR).

    Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Sequencing

    Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. (A) Volcano plot showing global translational changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Journal: bioRxiv

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    doi: 10.1101/2021.03.03.433675

    Figure Lengend Snippet: Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. (A) Volcano plot showing global translational changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection

    SARS-CoV-2 infection of Calu-3 cells (A) TCID 50 measurements of virus titres, (B) qRT-PCR measurements of intracellular viral RNA represented by 2 -ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and (C) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). (D) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).

    Journal: bioRxiv

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    doi: 10.1101/2021.03.03.433675

    Figure Lengend Snippet: SARS-CoV-2 infection of Calu-3 cells (A) TCID 50 measurements of virus titres, (B) qRT-PCR measurements of intracellular viral RNA represented by 2 -ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and (C) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). (D) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).

    Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining

    Unstable genes are more sensitive to translation inhibition. (A) Volcano plot showing global translation efficiency (Riborex engine) changes of 6,878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 hours versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Journal: bioRxiv

    Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

    doi: 10.1101/2021.03.03.433675

    Figure Lengend Snippet: Unstable genes are more sensitive to translation inhibition. (A) Volcano plot showing global translation efficiency (Riborex engine) changes of 6,878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 hours versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

    Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Inhibition, Infection

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Journal: bioRxiv

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    doi: 10.1101/2020.08.19.258244

    Figure Lengend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

    Techniques: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Journal: bioRxiv

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    doi: 10.1101/2020.08.19.258244

    Figure Lengend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

    Techniques: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Journal: bioRxiv

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    doi: 10.1101/2020.08.19.258244

    Figure Lengend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

    Techniques: Binding Assay, Functional Assay, Derivative Assay