rabbit anti sars cov 2 nucleocapsid protein pab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti sars cov 2 nucleocapsid protein pab
    Rabbit Anti Sars Cov 2 Nucleocapsid Protein Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti sars cov 2 nucleocapsid protein pab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti sars cov 2 nucleocapsid protein pab
    Rabbit Anti Sars Cov 2 Nucleocapsid Protein Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 nucleocapsid  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sars cov 2 nucleocapsid
    Characterization of spike protein processing of human <t>SARS-CoV-2</t> isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.
    Sars Cov 2 Nucleocapsid, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission"

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2022.01.006

    Characterization of spike protein processing of human SARS-CoV-2 isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.
    Figure Legend Snippet: Characterization of spike protein processing of human SARS-CoV-2 isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Techniques Used: Generated, Western Blot, Isolation, Infection, Expressing

    The H655Y amino acid substitution enhances spike cleavage and viral growth (A) Spike polymorphisms present in the mink-adapted variants, early human SARS-CoV-2 New York (NY) isolates, WA1-655Y, and WA1 wild-type viruses. Wuhan1 is included as a reference. (B) Replication kinetics of early SARS-CoV-2 viruses in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of S protein from supernatants of Vero E6- and Vero-TMPRSS2-infected cells. Infections were performed at an MOI of 0.01, and viral supernatants were collected at 48 h post-infection (p.i.). Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D and E) Quantification of full-length and cleaved spike protein of the indicated viruses in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. (F) Replication kinetics of early SARS-CoV-2 viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (G) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding early SARS-CoV-2 virus per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (H) Quantification of full-length and cleaved spike protein of the indicated viruses in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.
    Figure Legend Snippet: The H655Y amino acid substitution enhances spike cleavage and viral growth (A) Spike polymorphisms present in the mink-adapted variants, early human SARS-CoV-2 New York (NY) isolates, WA1-655Y, and WA1 wild-type viruses. Wuhan1 is included as a reference. (B) Replication kinetics of early SARS-CoV-2 viruses in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of S protein from supernatants of Vero E6- and Vero-TMPRSS2-infected cells. Infections were performed at an MOI of 0.01, and viral supernatants were collected at 48 h post-infection (p.i.). Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D and E) Quantification of full-length and cleaved spike protein of the indicated viruses in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. (F) Replication kinetics of early SARS-CoV-2 viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (G) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding early SARS-CoV-2 virus per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (H) Quantification of full-length and cleaved spike protein of the indicated viruses in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Techniques Used: Plaque Assay, Infection, Western Blot, Expressing

    The 655Y spike polymorphism increases cell-cell fusion (A) Immunofluorescence of SARS-CoV-2 S and N protein localization in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (B) Schematic representation of the split-GFP fusion assay. (C) Quantification of cell-cell fusion represented as GFP expression produced by each spike variant over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SDs of 3 independent experiments. (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei were stained using DAPI.
    Figure Legend Snippet: The 655Y spike polymorphism increases cell-cell fusion (A) Immunofluorescence of SARS-CoV-2 S and N protein localization in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (B) Schematic representation of the split-GFP fusion assay. (C) Quantification of cell-cell fusion represented as GFP expression produced by each spike variant over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SDs of 3 independent experiments. (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei were stained using DAPI.

    Techniques Used: Immunofluorescence, Infection, Staining, Single Vesicle Fusion Assay, Expressing, Produced, Variant Assay, Cytometry

    The 655Y polymorphism prevails over the 655H in the transmission in vivo model (A) Ten 3-week-old female Syrian hamsters were placed in pairs. Only 1 hamster per cage was infected intranasally with a total of 10 5 pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a one-to-one ratio. Nasal washes were collected at day 2, 4, and 6 post-infection (p.i.). Lungs and nasal turbinates were harvested from direct infected (DI) and direct contact (DC) hamsters at day 5 and 7 p.i., respectively. (B) Body weight change of individual hamsters over time. (C) Viral titers of nasal washes expressed as PFU per milliliter. Shown are the medians with 95% confidence intervals (CI). Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). (D) Relative abundance of 655Y mutation in the RNA from nasal washes in the DI and DC hamsters. The y axis shows the percentage of 655Y polymorphism in the total good quality sequencing reads from each biological RNA sample, and the x axis indicates the day p.i. samples were collected. (E) Viral titers of lungs and nasal turbinates expressed as PFU per gram of tissue. Shown are the medians with 95% CI. Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). Titers of DI and DC hamsters are shown at day 5 and 7 p.i., respectively. (F and G) Proportion of hamsters with 655Y (blue) and H (green) in the nasal turbinates and lungs from DI and DC as confirmed by next generation sequencing. (H) Competition experiments between SARS-CoV-2 WA1 and WA1-655Y isolates in human pneumocyte-like cells at different ratios. Proportion of 655Y (blue) and H (green) expressed as percentage are shown for the input and after 24 and 48 h p.i. Shown are the medians of 3 independent experiments.
    Figure Legend Snippet: The 655Y polymorphism prevails over the 655H in the transmission in vivo model (A) Ten 3-week-old female Syrian hamsters were placed in pairs. Only 1 hamster per cage was infected intranasally with a total of 10 5 pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a one-to-one ratio. Nasal washes were collected at day 2, 4, and 6 post-infection (p.i.). Lungs and nasal turbinates were harvested from direct infected (DI) and direct contact (DC) hamsters at day 5 and 7 p.i., respectively. (B) Body weight change of individual hamsters over time. (C) Viral titers of nasal washes expressed as PFU per milliliter. Shown are the medians with 95% confidence intervals (CI). Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). (D) Relative abundance of 655Y mutation in the RNA from nasal washes in the DI and DC hamsters. The y axis shows the percentage of 655Y polymorphism in the total good quality sequencing reads from each biological RNA sample, and the x axis indicates the day p.i. samples were collected. (E) Viral titers of lungs and nasal turbinates expressed as PFU per gram of tissue. Shown are the medians with 95% CI. Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). Titers of DI and DC hamsters are shown at day 5 and 7 p.i., respectively. (F and G) Proportion of hamsters with 655Y (blue) and H (green) in the nasal turbinates and lungs from DI and DC as confirmed by next generation sequencing. (H) Competition experiments between SARS-CoV-2 WA1 and WA1-655Y isolates in human pneumocyte-like cells at different ratios. Proportion of 655Y (blue) and H (green) expressed as percentage are shown for the input and after 24 and 48 h p.i. Shown are the medians of 3 independent experiments.

    Techniques Used: Transmission Assay, In Vivo, Infection, MANN-WHITNEY, Mutagenesis, Sequencing, Next-Generation Sequencing

    Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution frequencies around the cleavage site region (655 to 701) from globally available data (2,072,987 sequences deposited in GISAID database as of 28 June 2021) were estimated. (A) The high prevalent mutations identified mapped onto the structure of the S glycoprotein. The model was generated by superposition of PDB: 6M0J and 7C2L ( <xref ref-type=Chi et al., 2020 ; Lan et al., 2020 ). One RBD in the up conformation (red) is bound with ACE2 receptor (pink). The N-terminal domain (NTD) is colored blue, the amino-acid substitutions are shown as gold spheres, and the furin cleavage loop (disordered and therefore missing in most atomic models) is flanked with cyan spheres. One spike protomer is shown in bold colors while the other two are colored white. A zoomed-in image of the region of interest and the sequence of the furin site loop is also shown. Amino acid residues of interest are highlighted in gold. (B) Time-calibrated phylogenetic tree of SARS-CoV-2 circulating variants illustrating the temporal distribution and phylogenetic relationships of the most prevalent S mutations along the S1/S2 region (highlighted in color). The phylogenetic tree was generated using NextStrain, and analysis was performed using a sample of 13,847 genomes focused on the most prevalent substitutions between S:655 and S:701 between February 2020 and June 2021 from GISAID database. (C–E) Frequency per clade of H655Y, P681H/R, and A701V spike polymorphisms. " title="Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution frequencies around the cleavage site region (655 to 701) from globally available data (2,072,987 sequences deposited in GISAID database as of 28 June 2021) were estimated. (A) The high prevalent mutations identified mapped onto the structure of the S glycoprotein. The model was generated by superposition of PDB: 6M0J and 7C2L ( Chi et al., 2020 ; Lan et al., 2020 ). One RBD in the up conformation (red) is bound with ACE2 receptor (pink). The N-terminal domain (NTD) is colored blue, the amino-acid substitutions are shown as gold spheres, and the furin cleavage loop (disordered and therefore missing in most atomic models) is flanked with cyan spheres. One spike protomer is shown in bold colors while the other two are colored white. A zoomed-in image of the region of interest and the sequence of the furin site loop is also shown. Amino acid residues of interest are highlighted in gold. (B) Time-calibrated phylogenetic tree of SARS-CoV-2 circulating variants illustrating the temporal distribution and phylogenetic relationships of the most prevalent S mutations along the S1/S2 region (highlighted in color). The phylogenetic tree was generated using NextStrain, and analysis was performed using a sample of 13,847 genomes focused on the most prevalent substitutions between S:655 and S:701 between February 2020 and June 2021 from GISAID database. (C–E) Frequency per clade of H655Y, P681H/R, and A701V spike polymorphisms.

    Techniques Used: Generated, Sequencing

    SARS-CoV-2 VOCs evolve to a convergent phenotype associated to an increase on S cleavage (A) Multiple alignments of the S protein of the indicated SARS-CoV-2 VOCs. Diagram shows the corresponding S amino acid substitutions mapped to the S gene. (B) Viral growth of SARS-CoV-2 variants in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection (p.i.) and expressed as PFU per milliliter Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of spike cleavage in supernatants from Vero E6- and Vero-TMPRSS2-infected cells at an MOI of 0.01. Viral supernatants were collected at 48 h p.i. Full-length (FL) S protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D) Quantification of full-length and cleaved spike protein of the VOCs in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. Asterisk ( ∗ ) indicates under limit of detection. (E) Replication kinetics of SARS-CoV-2 VOCs in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (F) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding VOC per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (G) Quantification of full-length and cleaved spike protein of the VOCs in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression. (H) Quantification of the cleavage efficiency by mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cells extracts were collected after 24 h p.i. Cleavage efficiency was determined by measuring the abundance of the resulting peptide (SVASQSIIAYTMSLGAE) after cleavage at the terminal arginine of the furin cleavage site. Total spike, ORF3a, and N protein were used as internal standard to normalize across variants. The y axis shows the log 2 of fold change between cleaved peptide abundance for each variant normalized by WA1 control.
    Figure Legend Snippet: SARS-CoV-2 VOCs evolve to a convergent phenotype associated to an increase on S cleavage (A) Multiple alignments of the S protein of the indicated SARS-CoV-2 VOCs. Diagram shows the corresponding S amino acid substitutions mapped to the S gene. (B) Viral growth of SARS-CoV-2 variants in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection (p.i.) and expressed as PFU per milliliter Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of spike cleavage in supernatants from Vero E6- and Vero-TMPRSS2-infected cells at an MOI of 0.01. Viral supernatants were collected at 48 h p.i. Full-length (FL) S protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D) Quantification of full-length and cleaved spike protein of the VOCs in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. Asterisk ( ∗ ) indicates under limit of detection. (E) Replication kinetics of SARS-CoV-2 VOCs in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (F) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding VOC per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (G) Quantification of full-length and cleaved spike protein of the VOCs in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression. (H) Quantification of the cleavage efficiency by mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cells extracts were collected after 24 h p.i. Cleavage efficiency was determined by measuring the abundance of the resulting peptide (SVASQSIIAYTMSLGAE) after cleavage at the terminal arginine of the furin cleavage site. Total spike, ORF3a, and N protein were used as internal standard to normalize across variants. The y axis shows the log 2 of fold change between cleaved peptide abundance for each variant normalized by WA1 control.

    Techniques Used: Plaque Assay, Infection, Western Blot, Expressing, Mass Spectrometry, Variant Assay

    SARS-CoV-2 VOCs exhibit enhanced cell-cell fusion (A and B) Immunofluorescence of SARS-CoV-2 S and N protein in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. for the indicated VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (C) Quantification of cell-cell fusion represented as GFP expression showed by each spike VOC over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SD of 3 independent experiments. Two-tailed t test was performed to compare mean differences between each VOC S and corresponding reverse mutant. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei was stained using DAPI.
    Figure Legend Snippet: SARS-CoV-2 VOCs exhibit enhanced cell-cell fusion (A and B) Immunofluorescence of SARS-CoV-2 S and N protein in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. for the indicated VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (C) Quantification of cell-cell fusion represented as GFP expression showed by each spike VOC over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SD of 3 independent experiments. Two-tailed t test was performed to compare mean differences between each VOC S and corresponding reverse mutant. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei was stained using DAPI.

    Techniques Used: Immunofluorescence, Infection, Staining, Expressing, Two Tailed Test, Mutagenesis, Cytometry

    nucleocapsid antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc nucleocapsid antibody
    Nucleocapsid Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleocapsid antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    sars cov 2 nucleocapsid  (Cell Signaling Technology Inc)


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  • 94

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    Cell Signaling Technology Inc sars cov 2 nucleocapsid
    Construction of the HBV W4P preS1-fused pcDNA3.3-RBD plasmid (W4P-RBD) as a candidate for <t>SARS-CoV-2.</t> (A) Design of pcDNA3.3-RBD and pcDNA3.3-W4P-RBD. The W4P region comprises 33 bp from the first site of the preS1 region of the HBV genome and encodes 11 amino acids. (B) The protein expression of SARS-CoV-2 RBD and W4P-conjugated RBD was detected by the Western blot assay. pcDNA3.3-RBD, pcDNA3.3-W4P-RBD, and empty pcDNA3.3 were transfected into Vero E6, Huh7, and 293T cells, and cell lysates were collected 48 h post transfection to detect protein expression. (C) The mRNA expression levels of IL-6 and in pcDNA3.3-transfected cells were detected by qRT-PCR. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of three independent experiments.
    Sars Cov 2 Nucleocapsid, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 nucleocapsid/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    1) Product Images from "A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation"

    Article Title: A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.637654

    Construction of the HBV W4P preS1-fused pcDNA3.3-RBD plasmid (W4P-RBD) as a candidate for SARS-CoV-2. (A) Design of pcDNA3.3-RBD and pcDNA3.3-W4P-RBD. The W4P region comprises 33 bp from the first site of the preS1 region of the HBV genome and encodes 11 amino acids. (B) The protein expression of SARS-CoV-2 RBD and W4P-conjugated RBD was detected by the Western blot assay. pcDNA3.3-RBD, pcDNA3.3-W4P-RBD, and empty pcDNA3.3 were transfected into Vero E6, Huh7, and 293T cells, and cell lysates were collected 48 h post transfection to detect protein expression. (C) The mRNA expression levels of IL-6 and in pcDNA3.3-transfected cells were detected by qRT-PCR. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of three independent experiments.
    Figure Legend Snippet: Construction of the HBV W4P preS1-fused pcDNA3.3-RBD plasmid (W4P-RBD) as a candidate for SARS-CoV-2. (A) Design of pcDNA3.3-RBD and pcDNA3.3-W4P-RBD. The W4P region comprises 33 bp from the first site of the preS1 region of the HBV genome and encodes 11 amino acids. (B) The protein expression of SARS-CoV-2 RBD and W4P-conjugated RBD was detected by the Western blot assay. pcDNA3.3-RBD, pcDNA3.3-W4P-RBD, and empty pcDNA3.3 were transfected into Vero E6, Huh7, and 293T cells, and cell lysates were collected 48 h post transfection to detect protein expression. (C) The mRNA expression levels of IL-6 and in pcDNA3.3-transfected cells were detected by qRT-PCR. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of three independent experiments.

    Techniques Used: Plasmid Preparation, Expressing, Western Blot, Transfection, Quantitative RT-PCR

    W4P-RBD elicits RBD-specific antibody responses in serum and induces potent neutralizing activity against pseudotyped SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. (A,C) Antibody responses in serum specific to SARS-CoV-2 RBD proteins were detected by ELISA. (B) Serum at 5 weeks after the last immunization was assessed using different dilution factors for IgG against the SARS-CoV-2 RBD protein using ELISA. (D) The 50% neutralizing antibody titer (NT 50 ) was calculated using the SARS-CoV-2 pseudovirus neutralization assay in Calu-3 cells. (E) Correlation between SARS-CoV-2 RBD-specific IgG and pseudotyped SARS-CoV-2 neutralization titers for immunized mice. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.
    Figure Legend Snippet: W4P-RBD elicits RBD-specific antibody responses in serum and induces potent neutralizing activity against pseudotyped SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. (A,C) Antibody responses in serum specific to SARS-CoV-2 RBD proteins were detected by ELISA. (B) Serum at 5 weeks after the last immunization was assessed using different dilution factors for IgG against the SARS-CoV-2 RBD protein using ELISA. (D) The 50% neutralizing antibody titer (NT 50 ) was calculated using the SARS-CoV-2 pseudovirus neutralization assay in Calu-3 cells. (E) Correlation between SARS-CoV-2 RBD-specific IgG and pseudotyped SARS-CoV-2 neutralization titers for immunized mice. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    W4P-RBD exerts potent neutralizing activity against live SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) A 50% plaque reduction neutralizing antibody (PRNT 50 ) titer against live SARS-CoV-2 was calculated against SARS-CoV-2 infection in Vero E6 cells. (B) Reduction in plaque formation in Vero E6 cells infected with SARS-CoV-2. (C) Correlation between SARS-CoV-2 RBD-specific IgG and SARS-CoV-2 neutralization titers in immunized mice. Significance differences (** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.
    Figure Legend Snippet: W4P-RBD exerts potent neutralizing activity against live SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) A 50% plaque reduction neutralizing antibody (PRNT 50 ) titer against live SARS-CoV-2 was calculated against SARS-CoV-2 infection in Vero E6 cells. (B) Reduction in plaque formation in Vero E6 cells infected with SARS-CoV-2. (C) Correlation between SARS-CoV-2 RBD-specific IgG and SARS-CoV-2 neutralization titers in immunized mice. Significance differences (** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.

    Techniques Used: Activity Assay, Incubation, Neutralization, Infection

    The W4P-RBD vaccine exerts potent neutralizing activity against live SARS-CoV-2 and inhibits viral infection and replication of SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) The neutralization efficacy of serum from immunized mice against live SARS-CoV-2 RNA copy number in Vero E6 cells was determined by qRT-PCR. (B) The neutralization efficacy of the diluted serum against live SARS-CoV-2 in Vero E6 cells was determined by Western blotting. (C) Pooled serum (diluted 1:500) from each mouse group was tested in the neutralization assay against live SARS-CoV-2. Representative images (40-fold magnification) show live SARS-CoV-2-infected cells after neutralization in each group. Significance differences (* P < 0.05) among the different groups are shown in the related figures, and the RNA data are presented as the means ± s.e.m. of mice ( n = 7).
    Figure Legend Snippet: The W4P-RBD vaccine exerts potent neutralizing activity against live SARS-CoV-2 and inhibits viral infection and replication of SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) The neutralization efficacy of serum from immunized mice against live SARS-CoV-2 RNA copy number in Vero E6 cells was determined by qRT-PCR. (B) The neutralization efficacy of the diluted serum against live SARS-CoV-2 in Vero E6 cells was determined by Western blotting. (C) Pooled serum (diluted 1:500) from each mouse group was tested in the neutralization assay against live SARS-CoV-2. Representative images (40-fold magnification) show live SARS-CoV-2-infected cells after neutralization in each group. Significance differences (* P < 0.05) among the different groups are shown in the related figures, and the RNA data are presented as the means ± s.e.m. of mice ( n = 7).

    Techniques Used: Activity Assay, Infection, Incubation, Neutralization, Quantitative RT-PCR, Western Blot

    W4P-RBD potentiates functional T cells specific to SARS-CoV-2 S1 proteins. C57BL/6 mice were intramuscularly injected with W-RBD, W4P-RBD (50 μ g /mouse), or mock, and the spleens were collected 5 weeks post-vaccination for analysis by flow cytometry. (A,B) Splenocytes were incubated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 24 h and stained to detect IFNγ-producing CD8 + T cells and CD4 + T cells. (C) Correlation between RBD-specific IgG in serum and the S1-specific T-cell population in splenocytes. Significance differences (* P < 0.05, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.
    Figure Legend Snippet: W4P-RBD potentiates functional T cells specific to SARS-CoV-2 S1 proteins. C57BL/6 mice were intramuscularly injected with W-RBD, W4P-RBD (50 μ g /mouse), or mock, and the spleens were collected 5 weeks post-vaccination for analysis by flow cytometry. (A,B) Splenocytes were incubated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 24 h and stained to detect IFNγ-producing CD8 + T cells and CD4 + T cells. (C) Correlation between RBD-specific IgG in serum and the S1-specific T-cell population in splenocytes. Significance differences (* P < 0.05, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7). Pearson's correlations were calculated to define correlations.

    Techniques Used: Functional Assay, Injection, Flow Cytometry, Incubation, Staining

    W4P-RBD induces proinflammatory cytokine production in S1-stimulated splenocytes. Splenocytes from immunized mice were stimulated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 5 days. The cytokine production of (A) TNFα, (B) IFNγ, (C) IL-12p40, (D) IFNβ, (E) IL-6, and (F) IL-2 from splenocytes was detected by ELISA. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7).
    Figure Legend Snippet: W4P-RBD induces proinflammatory cytokine production in S1-stimulated splenocytes. Splenocytes from immunized mice were stimulated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 5 days. The cytokine production of (A) TNFα, (B) IFNγ, (C) IL-12p40, (D) IFNβ, (E) IL-6, and (F) IL-2 from splenocytes was detected by ELISA. Significance differences (* P < 0.05, ** P < 0.01, *** P < 0.001) among the different groups are shown in the related figures, and the data are presented as the means ± s.e.m. of mice ( n = 7).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Schematic representation of W4P-RBD as a vaccine candidate against SARS-CoV-2. A novel platform of N-terminal addition of HBV W4P preS1 33-bp sequences for a DNA vaccine against SARS-CoV-2 were developed. The W4P-RBD led to enhanced both humoral and cell-mediated immune response against SARS-CoV-2 in vaccinated mice, demonstrating its feasibility as a DNA vaccine to protect against SARS-CoV-2.
    Figure Legend Snippet: Schematic representation of W4P-RBD as a vaccine candidate against SARS-CoV-2. A novel platform of N-terminal addition of HBV W4P preS1 33-bp sequences for a DNA vaccine against SARS-CoV-2 were developed. The W4P-RBD led to enhanced both humoral and cell-mediated immune response against SARS-CoV-2 in vaccinated mice, demonstrating its feasibility as a DNA vaccine to protect against SARS-CoV-2.

    Techniques Used:

    proteins anti cox 2 rabbit polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc proteins anti cox 2 rabbit polyclonal antibody
    ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding <t>cox-2</t> or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.
    Proteins Anti Cox 2 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteins anti cox 2 rabbit polyclonal antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteins anti cox 2 rabbit polyclonal antibody - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability"

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    Journal: Scientific Reports

    doi: 10.1038/srep27275

    ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding cox-2 or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.
    Figure Legend Snippet: ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding cox-2 or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.

    Techniques Used: Incubation, Infection, Titration, Transfection, Plasmid Preparation, Over Expression, Expressing

    ( a,b ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) and PR8 (right panel) for the indicated times post infection (h p.i.). ( a ) Total cellular protein extracts were subjected to WB. One representative result of n = 3 is shown. ( b ) Cellular RNA from infected cells was extracted, reverse transcribed and analysed by quantitative real-time PCR (qRT-PCR). Expressional changes of COX-2 mRNA were normalised to 0 h as n-fold induction. Results are depicted as mean n-fold (±s.d.) of n = 3. Statistical significance was determined using one-way ANOVA followed by a Dunnett’s test. *P = 0.0102, ***P = 0.0003.
    Figure Legend Snippet: ( a,b ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) and PR8 (right panel) for the indicated times post infection (h p.i.). ( a ) Total cellular protein extracts were subjected to WB. One representative result of n = 3 is shown. ( b ) Cellular RNA from infected cells was extracted, reverse transcribed and analysed by quantitative real-time PCR (qRT-PCR). Expressional changes of COX-2 mRNA were normalised to 0 h as n-fold induction. Results are depicted as mean n-fold (±s.d.) of n = 3. Statistical significance was determined using one-way ANOVA followed by a Dunnett’s test. *P = 0.0102, ***P = 0.0003.

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

    ( a,b ) A549 cells were transfected with 1 μg RNA, extracted from IAV-infected A549 cells (vRNA), for indicated times post transfection (h p.t.). ( a ) Cellular RNA from transfected cells was extracted, reverse transcribed and analysed by qRT-PCR. Expressional changes of COX-2 mRNA were normalised to 0 h p.t. Results are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by a Dunnett’s test. ( a ) 2 h, P = 0.0188, 4 h, P = 0.0014, 6 h, P = 0.0047 and 8 h, P = 0.0035. ( b ) Total cellular protein extracts were analysed by WB. ( c ) 1 μg vRNA was dephosphorylated by phosphatase treatment and transfected into A549 cells for 6 h. Mock RNA and vRNA without phosphatase treatment were used as controls. Total cellular protein extracts were analysed by WB. ( d ) A549 cells were transfected with 1 μg of pCAGGS vector encoding for rig-i or empty vector as control for 24 h. Then cells were transfected with 1 μg vRNA or mock RNA for 4 h and COX-2 mRNA expression was determined via qRT-PCR as n-fold induction of empty vector mock RNA control. Results represent mean (±s.d.) of n = 3. ( e ) COX-2 mRNA expression after siRNA-mediated knock-down of RIG-I (48 h p.t.) and subsequent transfection of 1 μg vRNA or mock RNA for 4 h was determined via qRT-PCR. A negative siRNA served as control (siRNA control). Results are normalised to mock RNA control as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by a Sidak’s test ( d,e ). ( d ) *P = 0.0264 and ( e ) **P = 0.0013.
    Figure Legend Snippet: ( a,b ) A549 cells were transfected with 1 μg RNA, extracted from IAV-infected A549 cells (vRNA), for indicated times post transfection (h p.t.). ( a ) Cellular RNA from transfected cells was extracted, reverse transcribed and analysed by qRT-PCR. Expressional changes of COX-2 mRNA were normalised to 0 h p.t. Results are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by a Dunnett’s test. ( a ) 2 h, P = 0.0188, 4 h, P = 0.0014, 6 h, P = 0.0047 and 8 h, P = 0.0035. ( b ) Total cellular protein extracts were analysed by WB. ( c ) 1 μg vRNA was dephosphorylated by phosphatase treatment and transfected into A549 cells for 6 h. Mock RNA and vRNA without phosphatase treatment were used as controls. Total cellular protein extracts were analysed by WB. ( d ) A549 cells were transfected with 1 μg of pCAGGS vector encoding for rig-i or empty vector as control for 24 h. Then cells were transfected with 1 μg vRNA or mock RNA for 4 h and COX-2 mRNA expression was determined via qRT-PCR as n-fold induction of empty vector mock RNA control. Results represent mean (±s.d.) of n = 3. ( e ) COX-2 mRNA expression after siRNA-mediated knock-down of RIG-I (48 h p.t.) and subsequent transfection of 1 μg vRNA or mock RNA for 4 h was determined via qRT-PCR. A negative siRNA served as control (siRNA control). Results are normalised to mock RNA control as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by a Sidak’s test ( d,e ). ( d ) *P = 0.0264 and ( e ) **P = 0.0013.

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

    ( a,b ) A549 cells were infected with 1 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ). Total cellular protein extracts were produced 0–24 h p.i. and analysed by WB. ( c ) COX-2 mRNA synthesis in A549 cells was determined 8 h and 24 h p.i. with 1 MOI FPV by qRT-PCR. The results are normalised to the mock control and are depicted as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ***P = 0.0009. ( d ) Media, containing IAV virions of the subtype FPV, were treated with UV light to inactivate the virus. A549 cells were infected with UV-inactivated FPV or replicating virus for 18 h. Un-infected cells were used as control. Total cellular protein extracts were analysed by WB.
    Figure Legend Snippet: ( a,b ) A549 cells were infected with 1 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ). Total cellular protein extracts were produced 0–24 h p.i. and analysed by WB. ( c ) COX-2 mRNA synthesis in A549 cells was determined 8 h and 24 h p.i. with 1 MOI FPV by qRT-PCR. The results are normalised to the mock control and are depicted as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ***P = 0.0009. ( d ) Media, containing IAV virions of the subtype FPV, were treated with UV light to inactivate the virus. A549 cells were infected with UV-inactivated FPV or replicating virus for 18 h. Un-infected cells were used as control. Total cellular protein extracts were analysed by WB.

    Techniques Used: Infection, Produced, Quantitative RT-PCR

    ( a–d ) COX-2 mRNA stability was analysed by inhibition of mRNA synthesis with actinomycin D (ActD). A549 cells were infected with 5 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ) or transfected with 1 μg RNA extracted from IAV-infected (vRNA) or un-infected (mock RNA) A549 cells ( c ) or treated with 1 μg LPS ( d ) for 4 h. Subsequently, cells were treated with 3 μg ActD for 1 h or 2 h. After the times indicated post ActD treatment total RNA was isolated and subjected to qRT-PCR analysis for COX-2 mRNA. The n-fold induction of different treatments (0 h) was arbitrarily set to 100% and the other results were normalised to the respective 0 h time point. Data represent the mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. **P = 0.0068, ****P < 0.0001.
    Figure Legend Snippet: ( a–d ) COX-2 mRNA stability was analysed by inhibition of mRNA synthesis with actinomycin D (ActD). A549 cells were infected with 5 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ) or transfected with 1 μg RNA extracted from IAV-infected (vRNA) or un-infected (mock RNA) A549 cells ( c ) or treated with 1 μg LPS ( d ) for 4 h. Subsequently, cells were treated with 3 μg ActD for 1 h or 2 h. After the times indicated post ActD treatment total RNA was isolated and subjected to qRT-PCR analysis for COX-2 mRNA. The n-fold induction of different treatments (0 h) was arbitrarily set to 100% and the other results were normalised to the respective 0 h time point. Data represent the mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. **P = 0.0068, ****P < 0.0001.

    Techniques Used: Inhibition, Infection, Transfection, Isolation, Quantitative RT-PCR

    ( a ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) or PR8 (right panel). After indicated times p.i. RNA from cell lysates was isolated and reverse transcribed. TTP mRNA synthesis was analysed by qRT-PCR. The results were normalised to the time point 0 h and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by Dunnett’s test. FPV, *P = 0.0129, PR8, **P = 0.0091. ( b,c,e ) For knock-down of TTP A549 cells were transfected with 10 μM TTP siRNA or a negative control (siRNA control). After 48 h the transfected cells were infected with 5 MOI of FPV ( c ) or 5 MOI of PR8 ( b,c ), or 1 MOI of FPV or PR8 ( e ). ( b ) 8 h p.i. COX-2 mRNA synthesis was analysed by qRT-PCR. The results were normalised to mock-infected control and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. *P = 0.0100, ***P = 0.0004. ( c ) 8 h p.i. total cellular protein extracts were analysed by WB. ( d,f ) A549 cells were transfected with 1 μg of p3XFLAG-CMV-7.1 vector encoding murine ttp or the empty vector as control for 24 h. Subsequently, cells were infected with 5 MOI of FPV or PR8 ( d ), or 1 MOI of FPV or PR8 ( f ). ( d ) Total cellular protein extracts were analysed by WB 8 h p.i. ( e,f ) Viral titres were determined 24 h p.i. by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of siRNA control ( e ) or empty vector control ( f ) (n = 3). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( e ) FPV, *P = 0.0397, and PR8, **P = 0.0024, and ( f ) FPV, *P = 0.0111.
    Figure Legend Snippet: ( a ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) or PR8 (right panel). After indicated times p.i. RNA from cell lysates was isolated and reverse transcribed. TTP mRNA synthesis was analysed by qRT-PCR. The results were normalised to the time point 0 h and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by Dunnett’s test. FPV, *P = 0.0129, PR8, **P = 0.0091. ( b,c,e ) For knock-down of TTP A549 cells were transfected with 10 μM TTP siRNA or a negative control (siRNA control). After 48 h the transfected cells were infected with 5 MOI of FPV ( c ) or 5 MOI of PR8 ( b,c ), or 1 MOI of FPV or PR8 ( e ). ( b ) 8 h p.i. COX-2 mRNA synthesis was analysed by qRT-PCR. The results were normalised to mock-infected control and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. *P = 0.0100, ***P = 0.0004. ( c ) 8 h p.i. total cellular protein extracts were analysed by WB. ( d,f ) A549 cells were transfected with 1 μg of p3XFLAG-CMV-7.1 vector encoding murine ttp or the empty vector as control for 24 h. Subsequently, cells were infected with 5 MOI of FPV or PR8 ( d ), or 1 MOI of FPV or PR8 ( f ). ( d ) Total cellular protein extracts were analysed by WB 8 h p.i. ( e,f ) Viral titres were determined 24 h p.i. by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of siRNA control ( e ) or empty vector control ( f ) (n = 3). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( e ) FPV, *P = 0.0397, and PR8, **P = 0.0024, and ( f ) FPV, *P = 0.0111.

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Transfection, Negative Control, Plasmid Preparation, Titration

    IAV-induced activation of RIG-I initiates various signalling pathways resulting in the production of mediators as part of the inflammatory response including COX-2. In addition, IAV induces TTP via activation of RIG-I and mediates an inhibitory mechanism in which TTP promotes COX-2 mRNA degradation.
    Figure Legend Snippet: IAV-induced activation of RIG-I initiates various signalling pathways resulting in the production of mediators as part of the inflammatory response including COX-2. In addition, IAV induces TTP via activation of RIG-I and mediates an inhibitory mechanism in which TTP promotes COX-2 mRNA degradation.

    Techniques Used: Activation Assay

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    Characterization of spike protein processing of human <t>SARS-CoV-2</t> isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.
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    Characterization of spike protein processing of human <t>SARS-CoV-2</t> isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.
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    ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding <t>cox-2</t> or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.
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    ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding <t>cox-2</t> or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.
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    Characterization of spike protein processing of human SARS-CoV-2 isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: Characterization of spike protein processing of human SARS-CoV-2 isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Generated, Western Blot, Isolation, Infection, Expressing

    The H655Y amino acid substitution enhances spike cleavage and viral growth (A) Spike polymorphisms present in the mink-adapted variants, early human SARS-CoV-2 New York (NY) isolates, WA1-655Y, and WA1 wild-type viruses. Wuhan1 is included as a reference. (B) Replication kinetics of early SARS-CoV-2 viruses in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of S protein from supernatants of Vero E6- and Vero-TMPRSS2-infected cells. Infections were performed at an MOI of 0.01, and viral supernatants were collected at 48 h post-infection (p.i.). Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D and E) Quantification of full-length and cleaved spike protein of the indicated viruses in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. (F) Replication kinetics of early SARS-CoV-2 viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (G) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding early SARS-CoV-2 virus per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (H) Quantification of full-length and cleaved spike protein of the indicated viruses in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: The H655Y amino acid substitution enhances spike cleavage and viral growth (A) Spike polymorphisms present in the mink-adapted variants, early human SARS-CoV-2 New York (NY) isolates, WA1-655Y, and WA1 wild-type viruses. Wuhan1 is included as a reference. (B) Replication kinetics of early SARS-CoV-2 viruses in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of S protein from supernatants of Vero E6- and Vero-TMPRSS2-infected cells. Infections were performed at an MOI of 0.01, and viral supernatants were collected at 48 h post-infection (p.i.). Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D and E) Quantification of full-length and cleaved spike protein of the indicated viruses in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. (F) Replication kinetics of early SARS-CoV-2 viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (G) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding early SARS-CoV-2 virus per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (H) Quantification of full-length and cleaved spike protein of the indicated viruses in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Plaque Assay, Infection, Western Blot, Expressing

    The 655Y spike polymorphism increases cell-cell fusion (A) Immunofluorescence of SARS-CoV-2 S and N protein localization in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (B) Schematic representation of the split-GFP fusion assay. (C) Quantification of cell-cell fusion represented as GFP expression produced by each spike variant over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SDs of 3 independent experiments. (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei were stained using DAPI.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: The 655Y spike polymorphism increases cell-cell fusion (A) Immunofluorescence of SARS-CoV-2 S and N protein localization in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (B) Schematic representation of the split-GFP fusion assay. (C) Quantification of cell-cell fusion represented as GFP expression produced by each spike variant over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SDs of 3 independent experiments. (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei were stained using DAPI.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Immunofluorescence, Infection, Staining, Single Vesicle Fusion Assay, Expressing, Produced, Variant Assay, Cytometry

    The 655Y polymorphism prevails over the 655H in the transmission in vivo model (A) Ten 3-week-old female Syrian hamsters were placed in pairs. Only 1 hamster per cage was infected intranasally with a total of 10 5 pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a one-to-one ratio. Nasal washes were collected at day 2, 4, and 6 post-infection (p.i.). Lungs and nasal turbinates were harvested from direct infected (DI) and direct contact (DC) hamsters at day 5 and 7 p.i., respectively. (B) Body weight change of individual hamsters over time. (C) Viral titers of nasal washes expressed as PFU per milliliter. Shown are the medians with 95% confidence intervals (CI). Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). (D) Relative abundance of 655Y mutation in the RNA from nasal washes in the DI and DC hamsters. The y axis shows the percentage of 655Y polymorphism in the total good quality sequencing reads from each biological RNA sample, and the x axis indicates the day p.i. samples were collected. (E) Viral titers of lungs and nasal turbinates expressed as PFU per gram of tissue. Shown are the medians with 95% CI. Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). Titers of DI and DC hamsters are shown at day 5 and 7 p.i., respectively. (F and G) Proportion of hamsters with 655Y (blue) and H (green) in the nasal turbinates and lungs from DI and DC as confirmed by next generation sequencing. (H) Competition experiments between SARS-CoV-2 WA1 and WA1-655Y isolates in human pneumocyte-like cells at different ratios. Proportion of 655Y (blue) and H (green) expressed as percentage are shown for the input and after 24 and 48 h p.i. Shown are the medians of 3 independent experiments.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: The 655Y polymorphism prevails over the 655H in the transmission in vivo model (A) Ten 3-week-old female Syrian hamsters were placed in pairs. Only 1 hamster per cage was infected intranasally with a total of 10 5 pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a one-to-one ratio. Nasal washes were collected at day 2, 4, and 6 post-infection (p.i.). Lungs and nasal turbinates were harvested from direct infected (DI) and direct contact (DC) hamsters at day 5 and 7 p.i., respectively. (B) Body weight change of individual hamsters over time. (C) Viral titers of nasal washes expressed as PFU per milliliter. Shown are the medians with 95% confidence intervals (CI). Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). (D) Relative abundance of 655Y mutation in the RNA from nasal washes in the DI and DC hamsters. The y axis shows the percentage of 655Y polymorphism in the total good quality sequencing reads from each biological RNA sample, and the x axis indicates the day p.i. samples were collected. (E) Viral titers of lungs and nasal turbinates expressed as PFU per gram of tissue. Shown are the medians with 95% CI. Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). Titers of DI and DC hamsters are shown at day 5 and 7 p.i., respectively. (F and G) Proportion of hamsters with 655Y (blue) and H (green) in the nasal turbinates and lungs from DI and DC as confirmed by next generation sequencing. (H) Competition experiments between SARS-CoV-2 WA1 and WA1-655Y isolates in human pneumocyte-like cells at different ratios. Proportion of 655Y (blue) and H (green) expressed as percentage are shown for the input and after 24 and 48 h p.i. Shown are the medians of 3 independent experiments.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Transmission Assay, In Vivo, Infection, MANN-WHITNEY, Mutagenesis, Sequencing, Next-Generation Sequencing

    Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution frequencies around the cleavage site region (655 to 701) from globally available data (2,072,987 sequences deposited in GISAID database as of 28 June 2021) were estimated. (A) The high prevalent mutations identified mapped onto the structure of the S glycoprotein. The model was generated by superposition of PDB: 6M0J and 7C2L ( <xref ref-type=Chi et al., 2020 ; Lan et al., 2020 ). One RBD in the up conformation (red) is bound with ACE2 receptor (pink). The N-terminal domain (NTD) is colored blue, the amino-acid substitutions are shown as gold spheres, and the furin cleavage loop (disordered and therefore missing in most atomic models) is flanked with cyan spheres. One spike protomer is shown in bold colors while the other two are colored white. A zoomed-in image of the region of interest and the sequence of the furin site loop is also shown. Amino acid residues of interest are highlighted in gold. (B) Time-calibrated phylogenetic tree of SARS-CoV-2 circulating variants illustrating the temporal distribution and phylogenetic relationships of the most prevalent S mutations along the S1/S2 region (highlighted in color). The phylogenetic tree was generated using NextStrain, and analysis was performed using a sample of 13,847 genomes focused on the most prevalent substitutions between S:655 and S:701 between February 2020 and June 2021 from GISAID database. (C–E) Frequency per clade of H655Y, P681H/R, and A701V spike polymorphisms. " width="100%" height="100%">

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution frequencies around the cleavage site region (655 to 701) from globally available data (2,072,987 sequences deposited in GISAID database as of 28 June 2021) were estimated. (A) The high prevalent mutations identified mapped onto the structure of the S glycoprotein. The model was generated by superposition of PDB: 6M0J and 7C2L ( Chi et al., 2020 ; Lan et al., 2020 ). One RBD in the up conformation (red) is bound with ACE2 receptor (pink). The N-terminal domain (NTD) is colored blue, the amino-acid substitutions are shown as gold spheres, and the furin cleavage loop (disordered and therefore missing in most atomic models) is flanked with cyan spheres. One spike protomer is shown in bold colors while the other two are colored white. A zoomed-in image of the region of interest and the sequence of the furin site loop is also shown. Amino acid residues of interest are highlighted in gold. (B) Time-calibrated phylogenetic tree of SARS-CoV-2 circulating variants illustrating the temporal distribution and phylogenetic relationships of the most prevalent S mutations along the S1/S2 region (highlighted in color). The phylogenetic tree was generated using NextStrain, and analysis was performed using a sample of 13,847 genomes focused on the most prevalent substitutions between S:655 and S:701 between February 2020 and June 2021 from GISAID database. (C–E) Frequency per clade of H655Y, P681H/R, and A701V spike polymorphisms.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Generated, Sequencing

    SARS-CoV-2 VOCs evolve to a convergent phenotype associated to an increase on S cleavage (A) Multiple alignments of the S protein of the indicated SARS-CoV-2 VOCs. Diagram shows the corresponding S amino acid substitutions mapped to the S gene. (B) Viral growth of SARS-CoV-2 variants in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection (p.i.) and expressed as PFU per milliliter Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of spike cleavage in supernatants from Vero E6- and Vero-TMPRSS2-infected cells at an MOI of 0.01. Viral supernatants were collected at 48 h p.i. Full-length (FL) S protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D) Quantification of full-length and cleaved spike protein of the VOCs in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. Asterisk ( ∗ ) indicates under limit of detection. (E) Replication kinetics of SARS-CoV-2 VOCs in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (F) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding VOC per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (G) Quantification of full-length and cleaved spike protein of the VOCs in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression. (H) Quantification of the cleavage efficiency by mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cells extracts were collected after 24 h p.i. Cleavage efficiency was determined by measuring the abundance of the resulting peptide (SVASQSIIAYTMSLGAE) after cleavage at the terminal arginine of the furin cleavage site. Total spike, ORF3a, and N protein were used as internal standard to normalize across variants. The y axis shows the log 2 of fold change between cleaved peptide abundance for each variant normalized by WA1 control.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: SARS-CoV-2 VOCs evolve to a convergent phenotype associated to an increase on S cleavage (A) Multiple alignments of the S protein of the indicated SARS-CoV-2 VOCs. Diagram shows the corresponding S amino acid substitutions mapped to the S gene. (B) Viral growth of SARS-CoV-2 variants in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection (p.i.) and expressed as PFU per milliliter Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of spike cleavage in supernatants from Vero E6- and Vero-TMPRSS2-infected cells at an MOI of 0.01. Viral supernatants were collected at 48 h p.i. Full-length (FL) S protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D) Quantification of full-length and cleaved spike protein of the VOCs in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. Asterisk ( ∗ ) indicates under limit of detection. (E) Replication kinetics of SARS-CoV-2 VOCs in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (F) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding VOC per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (G) Quantification of full-length and cleaved spike protein of the VOCs in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression. (H) Quantification of the cleavage efficiency by mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cells extracts were collected after 24 h p.i. Cleavage efficiency was determined by measuring the abundance of the resulting peptide (SVASQSIIAYTMSLGAE) after cleavage at the terminal arginine of the furin cleavage site. Total spike, ORF3a, and N protein were used as internal standard to normalize across variants. The y axis shows the log 2 of fold change between cleaved peptide abundance for each variant normalized by WA1 control.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Plaque Assay, Infection, Western Blot, Expressing, Mass Spectrometry, Variant Assay

    SARS-CoV-2 VOCs exhibit enhanced cell-cell fusion (A and B) Immunofluorescence of SARS-CoV-2 S and N protein in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. for the indicated VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (C) Quantification of cell-cell fusion represented as GFP expression showed by each spike VOC over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SD of 3 independent experiments. Two-tailed t test was performed to compare mean differences between each VOC S and corresponding reverse mutant. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei was stained using DAPI.

    Journal: Cell Host & Microbe

    Article Title: Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

    doi: 10.1016/j.chom.2022.01.006

    Figure Lengend Snippet: SARS-CoV-2 VOCs exhibit enhanced cell-cell fusion (A and B) Immunofluorescence of SARS-CoV-2 S and N protein in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. for the indicated VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (C) Quantification of cell-cell fusion represented as GFP expression showed by each spike VOC over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SD of 3 independent experiments. Two-tailed t test was performed to compare mean differences between each VOC S and corresponding reverse mutant. Statistical significance was considered when p ≤ 0.05 ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant). (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei was stained using DAPI.

    Article Snippet: Anti-mouse secondary IgG-HRP antibody (Abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. β-actin was detected using IgG-HRP antibody (Cell Signaling; 5125) at dilution of 1:1000.

    Techniques: Immunofluorescence, Infection, Staining, Expressing, Two Tailed Test, Mutagenesis, Cytometry

    ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding cox-2 or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a,d ) A549 cells were pre-incubated either with 5 μM of CAY10404 or the solvent DMSO ( a ) or 5 μg/ml PGE2 or the solvent ethanol (EtOH) ( d ) for 1 h. Cells were infected with the IAV subtypes H7N7 (FPV; left panel) or H1N1 (PR8; right panel) at an MOI of 1 in addition to the treatment with CAY10404, PGE2 or the respective solvent for 24 h. Viral titres were determined by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of solvent control (n = 4) ( a ) or (n = 6) ( d ). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( a ) *P = 0.0337, and ( d ) **P = 0.0079, ****P < 0.0001. ( b,c ) A549 cells were transfected with 1 μg of pCMV-XL6 vector encoding cox-2 or the empty vector for 24 h. Cells were infected with 1 MOI of FPV or PR8 for 24 h. ( b ) Viral titres were determined by Standard Plaque Titration Assay as mean ( ± s.d.) in % of empty vector control (n = 5). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( b ) *P = 0.0107, **P = 0.0037. ( c ) Protein lysates were subjected to WB to analyse COX-2 overexpression and viral NS1 protein expression. Shown is one representative result of n = 3. ( e,f ) A549 were either pre-incubated with 5 μM CAY10404 ( e ) or 5 μg/ml PGE2 ( f ) or the respective solvent for 1 h prior to infection or were left un-treated. Cells were infected with FPV at an MOI of 0.01. Substances were added at the indicated times of infection. Viral titres were determined by Standard Plaque Titration Assay 10 h p.i. Results are depicted as mean (±s.d.) in % of solvent control of the indicated times of infection (n = 3). Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ( e ) −1 h *P = 0.0299, + 2 h *P = 0.0433, ***P = 0.0008, ****P < 0.0001, and ( f ) ***P = 0.0003, ****P < 0.0001.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

    Techniques: Incubation, Infection, Titration, Transfection, Plasmid Preparation, Over Expression, Expressing

    ( a,b ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) and PR8 (right panel) for the indicated times post infection (h p.i.). ( a ) Total cellular protein extracts were subjected to WB. One representative result of n = 3 is shown. ( b ) Cellular RNA from infected cells was extracted, reverse transcribed and analysed by quantitative real-time PCR (qRT-PCR). Expressional changes of COX-2 mRNA were normalised to 0 h as n-fold induction. Results are depicted as mean n-fold (±s.d.) of n = 3. Statistical significance was determined using one-way ANOVA followed by a Dunnett’s test. *P = 0.0102, ***P = 0.0003.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a,b ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) and PR8 (right panel) for the indicated times post infection (h p.i.). ( a ) Total cellular protein extracts were subjected to WB. One representative result of n = 3 is shown. ( b ) Cellular RNA from infected cells was extracted, reverse transcribed and analysed by quantitative real-time PCR (qRT-PCR). Expressional changes of COX-2 mRNA were normalised to 0 h as n-fold induction. Results are depicted as mean n-fold (±s.d.) of n = 3. Statistical significance was determined using one-way ANOVA followed by a Dunnett’s test. *P = 0.0102, ***P = 0.0003.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

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

    ( a,b ) A549 cells were transfected with 1 μg RNA, extracted from IAV-infected A549 cells (vRNA), for indicated times post transfection (h p.t.). ( a ) Cellular RNA from transfected cells was extracted, reverse transcribed and analysed by qRT-PCR. Expressional changes of COX-2 mRNA were normalised to 0 h p.t. Results are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by a Dunnett’s test. ( a ) 2 h, P = 0.0188, 4 h, P = 0.0014, 6 h, P = 0.0047 and 8 h, P = 0.0035. ( b ) Total cellular protein extracts were analysed by WB. ( c ) 1 μg vRNA was dephosphorylated by phosphatase treatment and transfected into A549 cells for 6 h. Mock RNA and vRNA without phosphatase treatment were used as controls. Total cellular protein extracts were analysed by WB. ( d ) A549 cells were transfected with 1 μg of pCAGGS vector encoding for rig-i or empty vector as control for 24 h. Then cells were transfected with 1 μg vRNA or mock RNA for 4 h and COX-2 mRNA expression was determined via qRT-PCR as n-fold induction of empty vector mock RNA control. Results represent mean (±s.d.) of n = 3. ( e ) COX-2 mRNA expression after siRNA-mediated knock-down of RIG-I (48 h p.t.) and subsequent transfection of 1 μg vRNA or mock RNA for 4 h was determined via qRT-PCR. A negative siRNA served as control (siRNA control). Results are normalised to mock RNA control as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by a Sidak’s test ( d,e ). ( d ) *P = 0.0264 and ( e ) **P = 0.0013.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a,b ) A549 cells were transfected with 1 μg RNA, extracted from IAV-infected A549 cells (vRNA), for indicated times post transfection (h p.t.). ( a ) Cellular RNA from transfected cells was extracted, reverse transcribed and analysed by qRT-PCR. Expressional changes of COX-2 mRNA were normalised to 0 h p.t. Results are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by a Dunnett’s test. ( a ) 2 h, P = 0.0188, 4 h, P = 0.0014, 6 h, P = 0.0047 and 8 h, P = 0.0035. ( b ) Total cellular protein extracts were analysed by WB. ( c ) 1 μg vRNA was dephosphorylated by phosphatase treatment and transfected into A549 cells for 6 h. Mock RNA and vRNA without phosphatase treatment were used as controls. Total cellular protein extracts were analysed by WB. ( d ) A549 cells were transfected with 1 μg of pCAGGS vector encoding for rig-i or empty vector as control for 24 h. Then cells were transfected with 1 μg vRNA or mock RNA for 4 h and COX-2 mRNA expression was determined via qRT-PCR as n-fold induction of empty vector mock RNA control. Results represent mean (±s.d.) of n = 3. ( e ) COX-2 mRNA expression after siRNA-mediated knock-down of RIG-I (48 h p.t.) and subsequent transfection of 1 μg vRNA or mock RNA for 4 h was determined via qRT-PCR. A negative siRNA served as control (siRNA control). Results are normalised to mock RNA control as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by a Sidak’s test ( d,e ). ( d ) *P = 0.0264 and ( e ) **P = 0.0013.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

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

    ( a,b ) A549 cells were infected with 1 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ). Total cellular protein extracts were produced 0–24 h p.i. and analysed by WB. ( c ) COX-2 mRNA synthesis in A549 cells was determined 8 h and 24 h p.i. with 1 MOI FPV by qRT-PCR. The results are normalised to the mock control and are depicted as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ***P = 0.0009. ( d ) Media, containing IAV virions of the subtype FPV, were treated with UV light to inactivate the virus. A549 cells were infected with UV-inactivated FPV or replicating virus for 18 h. Un-infected cells were used as control. Total cellular protein extracts were analysed by WB.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a,b ) A549 cells were infected with 1 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ). Total cellular protein extracts were produced 0–24 h p.i. and analysed by WB. ( c ) COX-2 mRNA synthesis in A549 cells was determined 8 h and 24 h p.i. with 1 MOI FPV by qRT-PCR. The results are normalised to the mock control and are depicted as mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA followed by Sidak’s test. ***P = 0.0009. ( d ) Media, containing IAV virions of the subtype FPV, were treated with UV light to inactivate the virus. A549 cells were infected with UV-inactivated FPV or replicating virus for 18 h. Un-infected cells were used as control. Total cellular protein extracts were analysed by WB.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

    Techniques: Infection, Produced, Quantitative RT-PCR

    ( a–d ) COX-2 mRNA stability was analysed by inhibition of mRNA synthesis with actinomycin D (ActD). A549 cells were infected with 5 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ) or transfected with 1 μg RNA extracted from IAV-infected (vRNA) or un-infected (mock RNA) A549 cells ( c ) or treated with 1 μg LPS ( d ) for 4 h. Subsequently, cells were treated with 3 μg ActD for 1 h or 2 h. After the times indicated post ActD treatment total RNA was isolated and subjected to qRT-PCR analysis for COX-2 mRNA. The n-fold induction of different treatments (0 h) was arbitrarily set to 100% and the other results were normalised to the respective 0 h time point. Data represent the mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. **P = 0.0068, ****P < 0.0001.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a–d ) COX-2 mRNA stability was analysed by inhibition of mRNA synthesis with actinomycin D (ActD). A549 cells were infected with 5 MOI of the IAV subtypes FPV ( a ) and PR8 ( b ) or transfected with 1 μg RNA extracted from IAV-infected (vRNA) or un-infected (mock RNA) A549 cells ( c ) or treated with 1 μg LPS ( d ) for 4 h. Subsequently, cells were treated with 3 μg ActD for 1 h or 2 h. After the times indicated post ActD treatment total RNA was isolated and subjected to qRT-PCR analysis for COX-2 mRNA. The n-fold induction of different treatments (0 h) was arbitrarily set to 100% and the other results were normalised to the respective 0 h time point. Data represent the mean (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. **P = 0.0068, ****P < 0.0001.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

    Techniques: Inhibition, Infection, Transfection, Isolation, Quantitative RT-PCR

    ( a ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) or PR8 (right panel). After indicated times p.i. RNA from cell lysates was isolated and reverse transcribed. TTP mRNA synthesis was analysed by qRT-PCR. The results were normalised to the time point 0 h and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by Dunnett’s test. FPV, *P = 0.0129, PR8, **P = 0.0091. ( b,c,e ) For knock-down of TTP A549 cells were transfected with 10 μM TTP siRNA or a negative control (siRNA control). After 48 h the transfected cells were infected with 5 MOI of FPV ( c ) or 5 MOI of PR8 ( b,c ), or 1 MOI of FPV or PR8 ( e ). ( b ) 8 h p.i. COX-2 mRNA synthesis was analysed by qRT-PCR. The results were normalised to mock-infected control and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. *P = 0.0100, ***P = 0.0004. ( c ) 8 h p.i. total cellular protein extracts were analysed by WB. ( d,f ) A549 cells were transfected with 1 μg of p3XFLAG-CMV-7.1 vector encoding murine ttp or the empty vector as control for 24 h. Subsequently, cells were infected with 5 MOI of FPV or PR8 ( d ), or 1 MOI of FPV or PR8 ( f ). ( d ) Total cellular protein extracts were analysed by WB 8 h p.i. ( e,f ) Viral titres were determined 24 h p.i. by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of siRNA control ( e ) or empty vector control ( f ) (n = 3). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( e ) FPV, *P = 0.0397, and PR8, **P = 0.0024, and ( f ) FPV, *P = 0.0111.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: ( a ) A549 cells were infected with 5 MOI of the IAV subtypes FPV (left panel) or PR8 (right panel). After indicated times p.i. RNA from cell lysates was isolated and reverse transcribed. TTP mRNA synthesis was analysed by qRT-PCR. The results were normalised to the time point 0 h and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA followed by Dunnett’s test. FPV, *P = 0.0129, PR8, **P = 0.0091. ( b,c,e ) For knock-down of TTP A549 cells were transfected with 10 μM TTP siRNA or a negative control (siRNA control). After 48 h the transfected cells were infected with 5 MOI of FPV ( c ) or 5 MOI of PR8 ( b,c ), or 1 MOI of FPV or PR8 ( e ). ( b ) 8 h p.i. COX-2 mRNA synthesis was analysed by qRT-PCR. The results were normalised to mock-infected control and are depicted as mean n-fold (±s.d.) of n = 3 independent experiments. Statistical significance was determined using two-way ANOVA followed by Sidak’s test. *P = 0.0100, ***P = 0.0004. ( c ) 8 h p.i. total cellular protein extracts were analysed by WB. ( d,f ) A549 cells were transfected with 1 μg of p3XFLAG-CMV-7.1 vector encoding murine ttp or the empty vector as control for 24 h. Subsequently, cells were infected with 5 MOI of FPV or PR8 ( d ), or 1 MOI of FPV or PR8 ( f ). ( d ) Total cellular protein extracts were analysed by WB 8 h p.i. ( e,f ) Viral titres were determined 24 h p.i. by Standard Plaque Titration Assay. Results are depicted as mean (±s.d.) in % of siRNA control ( e ) or empty vector control ( f ) (n = 3). Statistical significance was determined by using unpaired t-test with Welch’s correction. ( e ) FPV, *P = 0.0397, and PR8, **P = 0.0024, and ( f ) FPV, *P = 0.0111.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

    Techniques: Infection, Isolation, Quantitative RT-PCR, Transfection, Negative Control, Plasmid Preparation, Titration

    IAV-induced activation of RIG-I initiates various signalling pathways resulting in the production of mediators as part of the inflammatory response including COX-2. In addition, IAV induces TTP via activation of RIG-I and mediates an inhibitory mechanism in which TTP promotes COX-2 mRNA degradation.

    Journal: Scientific Reports

    Article Title: Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    doi: 10.1038/srep27275

    Figure Lengend Snippet: IAV-induced activation of RIG-I initiates various signalling pathways resulting in the production of mediators as part of the inflammatory response including COX-2. In addition, IAV induces TTP via activation of RIG-I and mediates an inhibitory mechanism in which TTP promotes COX-2 mRNA degradation.

    Article Snippet: For the detection of proteins anti-COX-2 rabbit polyclonal antibody from Cell Signaling Technology, anti-M1 mouse monoclonal antibody from Biorad, anti-NS1 mouse monoclonal antibody (clone NS1-23-1, developed at the Institute of Molecular Virology, Muenster, Germany), anti-α-Tubulin mouse monoclonal antibody and anti-flag M2 mouse monoclonal antibody from Sigma Aldrich, anti-COX-2 goat polyclonal antibody, anti-ERK2 (C-14) rabbit polyclonal antibody, anti-PB1 (vK-20) goat polyclonal antibody and anti-TTP (H-120) rabbit polyclonal antibody all from Santa Cruz Biotechnology, anti-phospho-STAT1 Tyr 701 (clone 14) mouse monoclonal antibody from BD Bioscience as well as anti-RIG-I mouse monoclonal antibody from Alexis Biochemicals (Enzo Life Sciences) were used.

    Techniques: Activation Assay