plex307 ace2 puro  (Addgene inc)

Journal: Lupus science & medicine

Article Title: SARS-CoV-2 spike aggravates lupus nephritis and lung fibrosis in systemic lupus erythematosus.

doi: 10.1136/lupus-2023-001104

Figure Lengend Snippet: Figure 1 COVID-19 spike protein increases autoantibody levels in the R848-induced BALB/c lupus model. (A) Plasmid map of pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro (Addgene). (B) BALB/c mice were treated with acetone (vehicle, n=5), and plasmids encoding blank (mock, n=5) or spike and ACE2 (spike, n=5) were injected into R848-induced lupus BALB/c mice. (C) Serum samples were harvested at the last time point and used for further analysis. Bar graph shows serum levels of antidouble stranded DNA (anti-dsDNA) total IgG, IgG1 and IgG2a in the indicated group. (D) Urine samples were harvested at the last time point and used for further analysis. Bar graph shows urine albumin levels in the indicated group. (E) Representative image showing spleen size in the indicated group. (F) Isolated peripheral blood cells were stained and subjected to flow cytometry. Bar graphs present the frequencies of interferon (IFN)-γ-positive, interleukin (IL)-4-positive and IL-17-positive cells in the blood. Data are means±SEMs (*p<0.05).

Article Snippet: Spike protein administration in the R848-induced BALB/c and IL-1Ra KO lupus model The plasmids mini- puro (158448; Addgene, Watertown, Massachusetts, USA) and pcDNA3.1- SARS2- SPIKE (145032; Addgene) were injected intravenously in 1 mL saline into R848- treated mice (spike group).

Techniques: Plasmid Preparation, Injection, Isolation, Staining, Flow Cytometry

Journal: Journal of inflammation (London, England)

Article Title: SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

doi: 10.1186/s12950-023-00362-x

Figure Lengend Snippet: Fig. 1 Overexpression of SARS-CoV-2 spike protein induces fibrotic effects in the HEK293 cell line. (a, b) Schematic representation and cloning site re gions of the pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro plasmids. (c) Western blotting analysis detecting the protein levels of fibrosis markers α-SMA and collagen type I with or without the overexpression of pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro in the HEK293 cell line. (d) Graph illustrating the levels of α-SMA and collagen type I with or without the overexpression of pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro in the HEK293 cell line. The data were expressed as the mean ± SEM for triplicate experiments, GAPDH was used as an internal control; *p < 0.05

Article Snippet: The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 μg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 μL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions.

Techniques: Over Expression, Cloning, Western Blot, Control

Journal: Journal of inflammation (London, England)

Article Title: SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

doi: 10.1186/s12950-023-00362-x

Figure Lengend Snippet: Fig. 2 Activation of the immune system in a BLM-induced SSc mouse model following overexpression of pcDNA3.1-SARS2-Spike and pLEX307-ACE2- puro (n = 5). (a) Experimental timeline depicting the schedule of pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro overexpression in the BLM-induced SSc mouse model. The mice received 100 µg/mL pcDNA3.1-SARS2-spike and 100 µg/mL pLEX307-ACE2-puro via intramuscular injection twice every 5 days and 0.5 mg/mL bleomycin via subcutaneous injection daily for 2 weeks. (b–d) Enzyme-linked immunoassay measurements of spike S protein, anti- phospholipid antibody, and IgG in serum from BLM-induced SSc mice over 35 days. (e) Flow cytometric analysis representing the percentage of ex vivo Th2 and Th17 cells from blood in BLM-induced SSc mice

Article Snippet: The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 μg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 μL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions.

Techniques: Activation Assay, Over Expression, Injection, Ex Vivo

Journal: Journal of inflammation (London, England)

Article Title: SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

doi: 10.1186/s12950-023-00362-x

Figure Lengend Snippet: Fig. 3 Accelerated fibrosis in BLM-induced SSc mice following injection of pcDNA3.1-SARS2-Spike and pLEX307-ACE2-puro (n = 5). (a) Confocal micros copy analysis showing CD4 (FITC), IL-17 (PE), and IL-4 (APC) staining. (b) Graph depicting the number of IL-4 + cells and IL-17 + cells among CD4 + positive cells. (c) Flow cytometric analysis illustrating the percentage of ex vivo Th2 and Th17 cells from the skin or lung in BLM-induced SSc mice

Article Snippet: The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 μg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 μL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions.

Techniques: Injection, Staining, Ex Vivo

Journal: Journal of inflammation (London, England)

Article Title: SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

doi: 10.1186/s12950-023-00362-x

Figure Lengend Snippet: Fig. 4 Induction of fibrosis in skin and lung tissues in BLM-Induced SSc mice following injection of pcDNA3.1-SARS2-Spike and pLEX307-ACE2-puro (n = 5). (a) Histological analysis of skin and lung tissues isolated from sacrificed SSc mouse models at 5 weeks post-injection, stained with hematoxylin & eosin, Masson’s trichrome, and subjected to immunohistochemistry analysis for α-SMA and collagen type I. (b) Graph representing skin dermal thickness, lung histological score, collagen area, α-SMA, and collagen type I positive area in skin and lung tissues

Article Snippet: The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 μg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 μL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions.

Techniques: Injection, Isolation, Staining, Immunohistochemistry

Journal: Journal of inflammation (London, England)

Article Title: SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

doi: 10.1186/s12950-023-00362-x

Figure Lengend Snippet: Fig. 5 Upregulation of inflammatory cytokines in BLM-induced SSc mice following injection of pcDNA3.1-SARS2-Spike and pLEX307-ACE2-puro (n = 5). (a) Immunohistochemical analysis of IL-6, IL-4, IL-17, TNFα, and IL-1β in skin and lung tissues isolated from sacrificed SSc mouse models at 5 weeks post- injection. (b) Graph illustrating the area positive for IL-6, IL-4, IL-17, TNFα, and IL-1β in skin and lung tissues

Article Snippet: The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 μg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 μL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions.

Techniques: Injection, Immunohistochemical staining, Isolation