Journal: MedComm

Article Title: Coronavirus M Protein Hijacks Toll‐Interacting Protein (TOLLIP) to Suppress NF‐κB Signaling and Promote Immune Evasion

doi: 10.1002/mco2.70821

Figure Lengend Snippet: SARS‐CoV‐2 M protein antagonized NF‐κB signaling in vivo. (A) The schematic illustration of experimental workflow in vivo. 6‐Week‐old C57BL/6 mice ( n = 6 per group) were intratracheal administration of AAV‐LungM3‐GFP (control vector) or AAV‐LungM3‐GFP‐M (M protein‐expressing vector) at a dose of 5 × 10 10 viral genomes (vg). At 21 days post‐AAV infection, pneumonia was induced via intranasal challenge with LPS (60 mg/kg), samples were collected at day 22. (B) Fluorescence microscopy analysis of GFP expression in lung tissues. Scale bars: 100 µm. (C) Immunoblot validation of GFP‐M fusion protein expression in major tissues (lung, heart, liver, intestine, kidney, and brain) of AAV‐GFP‐M‐infected mice. β‐Actin served as a loading control. (D) The histopathological changes of the lungs were examined by H&E staining. Scale bars: 100 µm. (E) Quantitative analysis of pathology scores of lung tissues (D). (F) RT‐qPCR analysis of proinflammatory gene expression ( Il1β , Il6 , Tnfα , Cxcl1 , and Cxcl10 ) in lung tissues 24 h after intranasal LPS administration. (G and H) Serum concentrations of proinflammatory cytokines IL‐1β (G) and IL‐6 (H) were measured by ELISA at 24 h post‐LPS challenge. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was evaluated using one‐way ANOVA for multiple groups (C) and two‐way ANOVA with Sidak's post hoc test (F‒H). p ‐Values for comparisons between the indicated groups are displayed in the figures.

Article Snippet: Briefly, mice were intratracheally injected with either AAV‐LungM3‐GFP‐M or AAV‐LungM3‐GFP (1 × 10 11 vg in 50 μL PBS per mouse; OBiO Technology).

Techniques: In Vivo, Control, Plasmid Preparation, Expressing, Infection, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Staining, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: MedComm

Article Title: Coronavirus M Protein Hijacks Toll‐Interacting Protein (TOLLIP) to Suppress NF‐κB Signaling and Promote Immune Evasion

doi: 10.1002/mco2.70821

Figure Lengend Snippet: SARS‐CoV‐2 M protein antagonized NF‐κB signaling in vivo. (A) The schematic illustration of experimental workflow in vivo. 6‐Week‐old C57BL/6 mice ( n = 6 per group) were intratracheal administration of AAV‐LungM3‐GFP (control vector) or AAV‐LungM3‐GFP‐M (M protein‐expressing vector) at a dose of 5 × 10 10 viral genomes (vg). At 21 days post‐AAV infection, pneumonia was induced via intranasal challenge with LPS (60 mg/kg), samples were collected at day 22. (B) Fluorescence microscopy analysis of GFP expression in lung tissues. Scale bars: 100 µm. (C) Immunoblot validation of GFP‐M fusion protein expression in major tissues (lung, heart, liver, intestine, kidney, and brain) of AAV‐GFP‐M‐infected mice. β‐Actin served as a loading control. (D) The histopathological changes of the lungs were examined by H&E staining. Scale bars: 100 µm. (E) Quantitative analysis of pathology scores of lung tissues (D). (F) RT‐qPCR analysis of proinflammatory gene expression ( Il1β , Il6 , Tnfα , Cxcl1 , and Cxcl10 ) in lung tissues 24 h after intranasal LPS administration. (G and H) Serum concentrations of proinflammatory cytokines IL‐1β (G) and IL‐6 (H) were measured by ELISA at 24 h post‐LPS challenge. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was evaluated using one‐way ANOVA for multiple groups (C) and two‐way ANOVA with Sidak's post hoc test (F‒H). p ‐Values for comparisons between the indicated groups are displayed in the figures.

Article Snippet: Briefly, mice were intratracheally injected with either AAV‐LungM3‐GFP‐M or AAV‐LungM3‐GFP (1 × 10 11 vg in 50 μL PBS per mouse; OBiO Technology).

Techniques: In Vivo, Control, Plasmid Preparation, Expressing, Infection, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Staining, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay