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 TRADD, FADD were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); antibody of TNFRSF1A was from Sigma.

Techniques: Control, Western Blot, Activity Assay, Immunoprecipitation, Incubation, Microscopy, Software

Journal: Scientific Reports

Article Title: METTL3-mediated m6A modification of TACSTD2 mRNA inhibits papillary thyroid cancer progression by activating the TNF signaling and blocking epithelial–mesenchymal transition

doi: 10.1038/s41598-025-30448-w

Figure Lengend Snippet: Knockdown of TACSTD2 promotes apoptosis by activating the TNF signaling pathway and blocking EMT to inhibit PTC progression. A : Difference volcano plot of KTC-1 cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#2, n = 3, |log2(FC)|> 1 and P < 0.05. B : KEGG analysis: TNF signaling pathway in PTC with TACSTD2-low expression was the top pathway based on mRNA-seq analysis. C–D : WB was used to measure TNF-α, TNF-R1, TNF-R2 and TRADD expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2. E : WB was used to measure Bax, Bcl-2, Caspase-3, cl-Caspase-3, Caspase-9 and cl-Caspase-9 expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2. F: WB was used to measure E-cadherin, N-cadherin, β-catenin, Snail, and Vimentin expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2.

Article Snippet: Equal protein amounts were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Germany), blocked with 5% non-fat milk at room temperature for 2 h prior to incubation overnight at 4 °C with primary antibodies against the following proteins: TACSTD2 (CST, #76730), METTL3 (CST, #86132), YTHDC2 (CST, #46324), MMP2 (CST, #40994), MMP3 (CST, #14351), MMP7 (CST, #3801), MMP9 (CST, #24317), TNF-α (CST, #11948), TNF-R1 (CST, #3736), TNF-R2 (CST, #72337), TRADD (CST, #3684), Bax (CST, #5023), Bcl-2 (CST, #4223), caspase-3 (CST, #9662), cleaved caspase-3 (CST, #9661), caspase-9, and cleaved caspase-9 (Proteintech, 10380-1-AP), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (Proteintech, 22018-1-AP), β-catenin (Proteintech, 51067-2-AP), Snail (CST, #3879), vimentin (CST, #5741), GAPDH (CST, #2118), and β-actin (CST, #4970).

Techniques: Knockdown, Blocking Assay, Transfection, Expressing