e protein e trunc  (ProSci Incorporated)

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 Ags induce macrophages to produce ROS and express proinflammatory chemokines and cytokines. (A and B) ROS production in peritoneal (A) and alveolar (B) macrophages after 20 µg/ml zymosan stimulation and incubation with SARS-CoV-2 peptides E-Trunc, S, and N. Representative figures for ROS production and area under the curve with n = 2 experiments: 6 biological and 6 technical for E-Trunc and N; n = 5 experiments: 15 biological and 15 technical for S for peritoneal macrophages; n = 2 experiments: 4 biological and 4 technical for E-Trunc and N; and n = 3 experiments: 6 biological and 6 technical for S for alveolar macrophages. (C and D) Detection of chemokines and cytokines in the culture supernatant of RAW (C) and THP1 (D) cells incubated with control, E-Trunc, or S at 2 µg/ml for 24 h. The graphs show measurements of the pixel density (n = 2 biological samples for each condition with 2 technical replicates per sample). (E) Expression of CCL5 and TNF-α RNA from THP1 cells incubated with control, S, or E-Trunc at 2 µg/ml for 3 h (n = 2 experiments: 4 biological and 4 technical replicates per sample). Graphs depict average with SEM. Mann–Whitney U test was used for statistical analysis in (A)–(D) and one-way ANOVA in (E). *p < 0.05, ****p < 0.0001. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Incubation, Expressing, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 Ags induce IFN and NF-κB signaling. (A) Fold change in IFN reporter activity in RAW, THP1, THP1-IRF3−/−, or THP1-TBK1−/− cells treated with control or polymyxin (Pb) at 10 µg/ml or SARS-CoV-2 Ags (E-Trunc, E-Full, S, or N) at 2 µg/ml and Pb at 10 µg/ml for 24 h (n = 2 experiments: 6 biological and 9 technical replicates for RAW with each viral Ag; n = 3 experiments: 9 biological and 6–12 technical replicates for THP1 with each viral Ag; and n = 2 experiments: 9 biological and 9 technical replicates for THP1-IRF3−/−and THP1-TBK1−/−cells with each viral Ag). (B) Fold change in NF-κB reporter activity in THP1 cells treated with control or Pb at 10 µg/ml or SARS-CoV-2 Ags (E-Trunc, E-Full, S, or N) at 2 µg/ml and Pb at 10 µg/ml for 24 h (n = 3 experiments: 9 biological and 6–12 technical replicates for THP1 with each viral Ag). Graphs depict average with SEM. *p < 0.05, ****p < 0.0001. ns, not statistically significant, by Mann–Whitney U test.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Activity Assay, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 viral Ags induce lung inflammation and vasculitis in mice. (A) Representative images of lung cross-sections from mice sacrificed 3 d after intranasal delivery of control, E-Trunc, or S at 10 µg. H&E-stained sections are shown. Boxed areas on the left are magnified adjacently. (B) Representative images of the lung cross-sections immunostained for CD45 expression. (C) Representative images of lung cross-sections depicting blood vessel pathology in each condition. Scale bars depicted in each picture. (D) Quantification of percentage of lobes with inflammatory infiltrates in lungs harvested in each condition (n = 3 mice per condition). (E) Representative images of the lung cross-sections stained for Isg15 by RNA in situ (n = 3 mice per condition). (F) Representative immunofluorescent images of the lung cross-sections immunostained for GFP and CD64 expression per above conditions; original magnification ×200 (n = 2 mice per condition). (G) Fold change in IFN reporter activity in A549 cells treated with control, Pb at 10 µg/ml, or SARS-CoV-2 Ags [2 µg/ml E-Trunc (i), E-Full (ii), S (iii), or N (iv)] and Pb at 10 µg/ml for 24 h (n = 2 experiments: 6 biological and 9–21 technical replicates for each viral Ag). Graphs depict average with SEM. One-way ANOVA in (D) and Mann–Whitney U test in (G) used for statistical analysis. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Staining, Expressing, In Situ, Activity Assay, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 viral Ag E induces airway epithelial IFN-dependent inflammation. (A) Representative images of lung cross-sections from Ifnar−/− mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 with 4–5 mice per condition). (B) Representative images of lung cross-sections from wt mice sacrificed 3 d after intranasal delivery of E-Trunc at 10 µg subsequent to Ifnar-depleting or isotype control Ab administration. H&E-stained sections are shown (n = 2 with 6 mice per condition). (C) Representative images of lung cross-sections from Ifnarf/f;LysM-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 mice per condition). (D) Representative images of lung cross-sections from Ifnarf/f;Cd11c-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 2 mice per condition). (E) Representative images of lung cross-sections from Ifnarf/f;Shh-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 with 8–9 mice per condition). Scale bars depicted in each picture. Severity scores per lobe are quantified to the right of each experimental condition in (A)–(E). Graphs depict average with SEM. ***p < 0.001, ****p < 0.0001, by Mann–Whitney U test. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Staining, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 Ags induce macrophages to produce ROS and express proinflammatory chemokines and cytokines. (A and B) ROS production in peritoneal (A) and alveolar (B) macrophages after 20 µg/ml zymosan stimulation and incubation with SARS-CoV-2 peptides E-Trunc, S, and N. Representative figures for ROS production and area under the curve with n = 2 experiments: 6 biological and 6 technical for E-Trunc and N; n = 5 experiments: 15 biological and 15 technical for S for peritoneal macrophages; n = 2 experiments: 4 biological and 4 technical for E-Trunc and N; and n = 3 experiments: 6 biological and 6 technical for S for alveolar macrophages. (C and D) Detection of chemokines and cytokines in the culture supernatant of RAW (C) and THP1 (D) cells incubated with control, E-Trunc, or S at 2 µg/ml for 24 h. The graphs show measurements of the pixel density (n = 2 biological samples for each condition with 2 technical replicates per sample). (E) Expression of CCL5 and TNF-α RNA from THP1 cells incubated with control, S, or E-Trunc at 2 µg/ml for 3 h (n = 2 experiments: 4 biological and 4 technical replicates per sample). Graphs depict average with SEM. Mann–Whitney U test was used for statistical analysis in (A)–(D) and one-way ANOVA in (E). *p < 0.05, ****p < 0.0001. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Incubation, Expressing, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 Ags induce IFN and NF-κB signaling. (A) Fold change in IFN reporter activity in RAW, THP1, THP1-IRF3−/−, or THP1-TBK1−/− cells treated with control or polymyxin (Pb) at 10 µg/ml or SARS-CoV-2 Ags (E-Trunc, E-Full, S, or N) at 2 µg/ml and Pb at 10 µg/ml for 24 h (n = 2 experiments: 6 biological and 9 technical replicates for RAW with each viral Ag; n = 3 experiments: 9 biological and 6–12 technical replicates for THP1 with each viral Ag; and n = 2 experiments: 9 biological and 9 technical replicates for THP1-IRF3−/−and THP1-TBK1−/−cells with each viral Ag). (B) Fold change in NF-κB reporter activity in THP1 cells treated with control or Pb at 10 µg/ml or SARS-CoV-2 Ags (E-Trunc, E-Full, S, or N) at 2 µg/ml and Pb at 10 µg/ml for 24 h (n = 3 experiments: 9 biological and 6–12 technical replicates for THP1 with each viral Ag). Graphs depict average with SEM. *p < 0.05, ****p < 0.0001. ns, not statistically significant, by Mann–Whitney U test.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Activity Assay, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 viral Ags induce lung inflammation and vasculitis in mice. (A) Representative images of lung cross-sections from mice sacrificed 3 d after intranasal delivery of control, E-Trunc, or S at 10 µg. H&E-stained sections are shown. Boxed areas on the left are magnified adjacently. (B) Representative images of the lung cross-sections immunostained for CD45 expression. (C) Representative images of lung cross-sections depicting blood vessel pathology in each condition. Scale bars depicted in each picture. (D) Quantification of percentage of lobes with inflammatory infiltrates in lungs harvested in each condition (n = 3 mice per condition). (E) Representative images of the lung cross-sections stained for Isg15 by RNA in situ (n = 3 mice per condition). (F) Representative immunofluorescent images of the lung cross-sections immunostained for GFP and CD64 expression per above conditions; original magnification ×200 (n = 2 mice per condition). (G) Fold change in IFN reporter activity in A549 cells treated with control, Pb at 10 µg/ml, or SARS-CoV-2 Ags [2 µg/ml E-Trunc (i), E-Full (ii), S (iii), or N (iv)] and Pb at 10 µg/ml for 24 h (n = 2 experiments: 6 biological and 9–21 technical replicates for each viral Ag). Graphs depict average with SEM. One-way ANOVA in (D) and Mann–Whitney U test in (G) used for statistical analysis. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Staining, Expressing, In Situ, Activity Assay, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Surface Proteins of SARS-CoV-2 Drive Airway Epithelial Cells to Induce IFN-Dependent Inflammation

doi: 10.4049/jimmunol.2001407

Figure Lengend Snippet: SARS-CoV-2 viral Ag E induces airway epithelial IFN-dependent inflammation. (A) Representative images of lung cross-sections from Ifnar−/− mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 with 4–5 mice per condition). (B) Representative images of lung cross-sections from wt mice sacrificed 3 d after intranasal delivery of E-Trunc at 10 µg subsequent to Ifnar-depleting or isotype control Ab administration. H&E-stained sections are shown (n = 2 with 6 mice per condition). (C) Representative images of lung cross-sections from Ifnarf/f;LysM-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 mice per condition). (D) Representative images of lung cross-sections from Ifnarf/f;Cd11c-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 2 mice per condition). (E) Representative images of lung cross-sections from Ifnarf/f;Shh-Cre(+/–) mice sacrificed 3 d after intranasal delivery of control or E-Trunc at 10 µg. H&E-stained sections are shown (n = 3 with 8–9 mice per condition). Scale bars depicted in each picture. Severity scores per lobe are quantified to the right of each experimental condition in (A)–(E). Graphs depict average with SEM. ***p < 0.001, ****p < 0.0001, by Mann–Whitney U test. ns, not statistically significant.

Article Snippet: Cells were seeded at 1 × 10 5 cells per 96 wells and incubated with SARS-CoV-2 S, E-Trunc, N protein (40588-V08B; Sino Biological), SARS-CoV-1 S (40634-V08B; Sino Biological), or MERS S (40069-V08B; Sino Biological) at 2 µg/ml or equal volume of water as control.

Techniques: Staining, MANN-WHITNEY