lentiviral vector plenti cytomegalovirus green fluorescent protein puro  (Addgene inc)

Journal: Frontiers in Pharmacology

Article Title: Licochalcone A suppresses pancreatic ductal adenocarcinoma progression by targeting eEF2K-mediated pyroptosis

doi: 10.3389/fphar.2025.1595686

Figure Lengend Snippet: LHA induces pyroptosis through targeting eEF2K in PDAC cells. (A) Molecular docking of LHA on eEF2K protein. Predicted two-dimensional and three-dimensional crystal structures of LHA (PubChem CID: 5318998) and the eEF2K (PDB ID: 7SHQ) complex. (B) MIA PaCa-2 and BxPc3 cell lines were treated with LHA (0, 7.5, 10, and 12.5 μM) for 48 h. The expressions of eEF2K, p-eEF2, and eEF2 were examined using Western blotting. Data were expressed as mean ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. (C) MIA PaCa-2 and BxPc3 cell lines were transfected using lentivirus vectors with the overexpressed human eEF2K gene, and the images were captured using the fluorescent microscope after 72 h of transfection. (D) MIA PaCa-2 and BxPc3 with or without eEF2K overexpression. The expressions of eEF2K were examined using Western blotting. Data were expressed as mean ± S.D. of at least three independent experiments. **p < 0.01 vs. control. (E) The cell viability of MIA PaCa-2 and BxPc3 cell lines with or without overexpression of eEF2K was examined using MTT assay. Data were expressed as mean ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. overexpression eEF2K with the same LHA concentration. (F) MIA PaCa-2 and BxPc3 with or without eEF2K overexpression with LHA (0 and 12.5 μM) for 48 h. The expressions of eEF2K, GSDMD-N, GSDME-N, and IL-1β were examined using Western blotting. Data were expressed as mean ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. ## p < 0.01 and ### p < 0.001 vs. overexpression eEF2K with the same LHA concentration.

Article Snippet: Lentivirus vectors carrying the overexpressed human eEF2K gene and green fluorescent protein (GFP) were designed and produced as “LV-EEF2K (100354-1).” The construct was bought from the Genchem Company (Shanghai, China).

Techniques: Western Blot, Control, Transfection, Microscopy, Over Expression, MTT Assay, Concentration Assay

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: TNIP3 overexpression improved cardiac function and fibrosis in ISO-induced heart failure mice. ( A ) Schematic diagram of experimental procedure in vivo study. ( B ) Representative echocardiogram images. ( C–D ) Quantitative of LVEF and LVFS ( n = 6). ( E–F ) Quantitative of LVESD and LVEDD ( n = 6). ( G ) Representative images of PSR-stained heart sections and quantitative of percentage of the fibrotic area ( n = 5 per group). ( G ) Quantitative of mRNA levels of collagen-I , collagen-III , and TGF-β ( n = 4 per group). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group. ISO, isoproterenol; LVEDD, left ventricular end-systolic diameter; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-diastolic diameter; LVFS, left ventricular fractional shortening; PSR, Picric-Sirius red.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression, In Vivo, Staining

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: TNIP3 overexpression diminished ventricular arrhythmia susceptibility in ISO-induced heart failure mice. ( A ) Representative electrocardiogram images. ( B–C ) Quantitative of QRS interval and QTc ( n = 6 per group). ( D ) Representative examples of VA induced by burst-stimulating. ( E ) Ratio of burst-induced VAs and ( F ) duration of VA ( n = 10 per group). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group. SR, sinus rhythm; VA, ventricular arrhythmia.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: TNIP3 overexpression reversed CV reduction in ISO-induced heart failure mice. ( A ) Typical examples and quantitative results of Cx43 immunofluorescence staining ( n = 5). ( B ) Representative western blot and statistical results of Cx43 protein expression ( n = 4). ( C ) Representative image of epicardial conduction recorded by optical mapping and quantitative results of CV ( n = 5). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group. CV, conduction velocity. Cx43, Connexin 43.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: TNIP3 overexpression reversed electrical remodelling in ISO-induced heart failure mice. ( A–E ) Representative western blot and statistical results of Cav1.2, Kv1.5, and Kv4.3 protein expression ( n = 4). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression, Western Blot, Expressing

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: TNIP3 overexpression alleviated inflammatory response in ISO-induced heart failure mice. ( A ) Heatmap of RNA-Seq expression levels of DEGS in ISO + AAV9-GFP vs. ISO + AAV9-TNIP3 ventricles ( n = 5 per group). ( B ) Gene Ontology (GO) enrichment analysis of the biological process. ( C ) Gene set enrichment analysis (GSEA). ( D ) Representative CD68, iNOS, and CD206 immunofluorescence and quantitative results of CD68, iNOS, and CD206-positive cells ( n = 6 per group). ( E ) Quantitative results of iNOS, IL-6, Arg1, and Mrc1 mRNA expression ( n = 4 per group). ( F ) Representative western blot and statistical results of protein levels of IL-1β, IL-6, and TNF-α ( n = 4 per group). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group. DEGs, differential expression genes; IL-1β, interleukin 1β; IL-6, interleukin 6; RNA-Seq, RNA sequencing; TNF-α, tumour necrosis factor α.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression, RNA Sequencing Assay, Expressing, Immunofluorescence, Western Blot

Journal: Europace

Article Title: Tumour necrosis factor alpha-induced protein 3-interacting protein 3 overexpression protects against arrhythmogenic remodelling in the heart failure mice

doi: 10.1093/europace/euaf002

Figure Lengend Snippet: PI3K/Akt/NF-κB signalling is responsible for the protective effect of TNIP3 overexpression on ISO-induced heart failure. ( A ) KEGG analysis. ( B ) GSEA of KEGG analysis. ( C ) Representative western blot and statistical results of protein levels of p-PI3K, PI3K, p-Akt, Akt, p-P65, and P65 ( n = 4 per group). * P < 0.05 vs. CTL + AAV9-GFP group. # P < 0.05 vs. ISO + AAV9-GFP group. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: To overexpress TNIP3 in mice, the mice were injected via the tail vein with adeno-associated viral 9 vector (AAV9) (Genechem Co., Ltd, Shanghai, China) carrying TNIP3 (AAV9-cTNT-TNIP3) or AAV9 vector that expressed green fluorescent protein (GFP) as control (AAV9-GFP).

Techniques: Over Expression, Western Blot