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Image Search Results
Journal: British Journal of Pharmacology
Article Title: 14-Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor-alpha-induced apoptosis through calcium-dependent tumour necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway
doi: 10.1111/j.1476-5381.2010.00836.x
Figure Lengend Snippet: 14-DAG inhibited TNF-α-mediated DISC formation. (A) Hepatocytes were pretreated with 14-DAG (5, 10 and 15 nM) for 1 h and then exposed to TNF-α/ActD for 12 h. Control cells were treated with the vehicle, DMSO. Immunoreactive bands of the active caspase-3 fragment were analysed by Western blot. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Caspase-3 activity was determined by using the fluorometric substrates DEVD–AFC. The data are shown as mean ± SD of three independent experiments. *P < 0.02, **P < 0.01, versus TNF-α/ActD-treated cells. (B) Caspase-8 activity was also determined by using the fluorometric substrates IETD–AFC in different treatment groups similar to caspase-3. The data are shown as mean ± SD of three independent experiments. *P < 0.02, **P < 0.01, versus TNF-α/ActD-treated cells. (C) Hepatocytes were pretreated with 14-DAG for 1 h at 37°C followed by treatment with TNF-α (10 ng·mL−1) and ActD (200 ng·mL−1) for 1 h at 37°C. TNFRSF1A was immunoprecipitated from the cell lysate, and the immunoprecipitates (IPs) were electrophoresed and immunoblotted with anti-FADD, anti-TRADD and anti-caspase-8 (p43) antibodies. Densitometric analyses of IPs were performed. The data are shown as mean ± SD of three independent experiments. (D) Hepatocytes were pretreated with 14-DAG for 1 h followed by incubation in PBS (pH 7.4) containing biotinylated TNF-α (10 ng·mL−1). TNFRSF1A internalization in hepatocytes was viewed under laser scanning confocal microscope using streptavidin–FITC. The magnification of the photomicrograph is 100×. (E) Evaluation of TNFRSF1A internalization in the presence of 5, 10 and 15 nM 14-DAG (experimental conditions were same as D) was quantified by FACS analysis using CELL Quest software. Results are representative of seven independent experiments with similar results.
Article Snippet: Fluorescent dyes were from Molecular Probes (Invitrogen Corporation); Ru360 was from Calbiochem (EMD Bioscience, Gibbstown, NJ, USA); 1,3,4,6-tetrachloro-3a, 6a di phenyl glycoluril (chloroglycoluril) (PIERCE, Rockford, IL, USA); antibodies of
Techniques: Control, Western Blot, Activity Assay, Immunoprecipitation, Incubation, Microscopy, Software