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

Journal: STAR Protocols

Article Title: Protocol for targeted gene manipulation and thermogenic evaluation in mouse brown adipocytes

doi: 10.1016/j.xpro.2025.104317

Figure Lengend Snippet: Assessment of Futile Creatine Cycle within mature brown adipocytes (A) Representative immunofluorescence images of iBAT transduced with AAV-FLEX-GFP-FLAG. Mature adipocytes were labelled with anti-Perilipin 1 (PLIN1) antibody (red), GFP-FLAG was labeled with anti-FLAG antibody (green). Nuclei were labelled with DAPI (blue). Scale bar, 100 μm. Figure adapted from figure 1d of Bunk et al. (B) Oxygen consumption rates basally and following sequential additions of noradrenaline (NA, 0.1 μM) and oligomycin (Oligo, 15 μM). SBI-425 (10 μM) was added at the start (n = 2 independent preparations/sex). Figure adapted from figure 5b of Bunk et al. Two-way ANOVA (Tukey’s post-hoc test). Data are represented as mean ± SEM.

Article Snippet: AAV GFP (plasmid) , Addgene , 49055.

Techniques: Immunofluorescence, Transduction, Labeling