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: PLoS ONE

Article Title: Electroacupuncture-Like Stimulation at the Baihui (GV20) and Dazhui (GV14) Acupoints Protects Rats against Subacute-Phase Cerebral Ischemia-Reperfusion Injuries by Reducing S100B-Mediated Neurotoxicity

doi: 10.1371/journal.pone.0091426

Figure Lengend Snippet: Representative western blot images show the expression of (A) mitochondrial cytochrome c and (C) cytosolic cytochrome c, TRADD, FADD, cleaved caspase-8 and cleaved caspase-3 in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups 7 d after reperfusion. COX IV and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively. The relative mitochondrial expression of (B) cytochrome c, and the relative cytosolic expression of (D) cytochrome c, (E) TRADD, (F) FADD, (G) cleaved caspase-8, and (H) cleaved caspase-3 were evaluated in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups (n = 4). Data are presented as mean ± SD. * P <0.05 compared with the Sham-7 d group; # P <0.05 compared with the Model-7 d group.

Article Snippet: They were then incubated with a mouse anti-GFAP (1∶1000 dilution, #3670 Cell Signaling Technology), rabbit anti-phospho-SAPK/JNK (p-JNK (Thr183/Tyr185); 1∶1000 dilution, #9251S Cell Signaling Technology), rabbit anti-phospho-p44/42 mitogen-activated protein kinase (MAPK (p-ERK); 1∶1000 dilution, #9101 Cell Signaling Technology), rabbit anti-phospho-p38 MAP kinase (p-p38 MAP kinase (Thr180/Tyr182); 1∶1000 dilution, #9212 Cell Signaling Technology), rabbit anti-cytochrome c (1∶1000 dilution, #4272 Cell Signaling Technology), rabbit anti-tumor necrosis factor receptor type 1-associated death domain (TRADD) (1∶1000 dilution, #3694 Cell Signaling Technology), rabbit anti-Fas-associated death domain (FADD) (1∶1000 dilution, #341282 Calbiochem), rabbit anti-cleaved caspase-8 (1∶1000 dilution, 3259-100 BioVision), or rabbit anti-cleaved caspase-3 (1∶1000 dilution, #9661S Cell Signaling Technology) antibody overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: Deficiency of TRADD induces impaired DNA damage response. ( a ) Western blotting analysis shows blotting for γH2AX, TRADD, and Actin in TRADD +/+ and TRADD −/− MEF cells treated with H 2 O 2 in time-dependent manner (0.5 mM). ( b ) γH2AX foci (Red) were analyzed in TRADD +/+ and TRADD −/− MEF cells treated with H 2 O 2 (0.5 mM) by Immunofluorescense as described in A. Scale bars, 10 μm. ( c ) Immunofluorescence analyses of γH2AX (Green) in H 2 O 2 (0.5 mM) treated TRADD +/+ and TRADD −/− MEF for 2 hours (upper panels) and release from H 2 O 2 treated TRADD +/+ and TRADD −/− MEF for 4 hours (lower panels). Cells were stained with anti-γH2AX (Green) and DAPI (Blue). Scale bars, 10 μm. ( d ) Western blotting analysis shows results consistent with immunofluorescence as described in ( c ). ( e ) After cells were treated with etoposide (25 μM) for 1 hour, TRADD +/+ and TRADD −/− MEF replaced with fresh media. Cells were stained with anti-γH2AX (Red) and DAPI (Blue). Western blotting analysis (lower panel) shows the consistent results with immunofluorescence. Scale bars, 10 μm. ( f ) Quantitative analysis of γH2AX foci was conducted in TRADD knock-downed U2OS cells. After TRADD knockdown, cells were treated with phleomycin (Phleo) and then stained with γH2AX antibody. *P < 0.05 (Student’ s t-test). ( g ) Transient knockdown of TRADD induces unrepaired DNA damage in HeLa cells. Western blot analysis shows γH2AX status in response to H 2 O 2 in TRADD KD HeLa cells. Cells were transfected with siRNA TRADD or siRNA negative control (NC), respectively. After 48 hours, the cells were continuously treated with H 2 O 2 (0.5 mM). The whole cell lysates were analysed by western blot as using indicated antibodies. ( h ) Reconstitution of TRADD in TRADD −/− MEFs. Western blotting analysis shows γH2AX expression in response to continuous treatment with H 2 O 2 (0.5 mM) in different time points.

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Expressing

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: DNA damage induces nuclear translocation of TRADD. ( a ) HeLa cells were transiently transfected with GFP-TRADD and treated with H 2 O 2 (0.5 mM) for indicated time points. Cells were analyzed by confocal fluorescence microscopy. ( b ) HeLa cells were transiently transfected with GFP-TRADD and treated with H 2 O 2 (0.5 mM). After treatment, live cell Images were analyzed by confocal fluorescence microscopy for 70 minutes (left panel). Quantitative analysis of nuclear translocation of TRADD was measured by GFP intensity in the nucleus (right panel). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (Student’s t-test). ( c ) Colocalization of GFP-TRADD and mCherry-FokI at single DNA double-strand break site. GFP empty vector (EV), GFP-TRADD wild type (WT), or GFP-TRADD Src mutant (SRC) was cotranfected with mCherry-FokI (mCh-FokI) nuclease into U2OS 2-6-3 cell lines. After 48 hr, cells were fixed and stained with DAPI for nuclear staining. Images were analyzed confocal microscope (Nikon A1). Scale bar, 10 μm. ( d ) HeLa cell were transiently transfected with NES mutant TRADD and treated with H 2 O 2 (0.5 mM) or MNNG (0.25 mM) for indicated times. Cells were fractionated into cytoplasmic and nuclear fractions using an NE-PER fractionation kit. Anti-Hsp90 or anti-Sp1 used as a control for normalization of cytoplasm and nuclear lysates, respectively. ( e ) Western blotting analysis was conducted with lysates from TRADD −/− (MOCK), NES-mutant TRADD (NES-TRADD) and Src-myristoylation-TRADD (Src-TRADD) in TRADD −/− MEFs treated with H 2 O 2 (0.5 mM) for indicated time periods (left panel). Expression of γH2AX was analyzed in TRADD −/− and TRADD −/− (NES-mutant TRADD) MEFs treated with H 2 O 2 (0.5 mM) for 1 hour using immunofluorescence (right panels).

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Translocation Assay, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Mutagenesis, Staining, Fractionation, Control, Western Blot, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

doi: 10.1038/s41598-017-03211-z

Figure Lengend Snippet: TRADD is required for non-homologous end-joining repair. ( a ) Knockdown efficacy for TRADD in DNA repair reporter cell lines EJ5 and DR. ( b ) After 48 hours transfection with TRADD, RPA80, or BRCA1 targeting siRNAs into reporter cell lines, each siRNA was again cotransfected with an I- SceI endonuclease construct. After 72 hours, GFP positive cells were analyzed with a flow cytometer (FACScan). **P < 0.01; ***P < 0.001; n.s., not significant (ANOVA). ( c , d ) After 1 hour with laser microirradiation, endogenous NHEJ repair factors were stained with each antibody at DNA break sites: 53BP1 ( c ); Ku70/80 and 53BP1 ( d ). Scale bars, 10 μm. ( e , f ) Endogenous HR repair factors were stained as described in c . RAD51 ( e ); RPA32 and RAD51 ( f ). γH2AX was used as a DNA damage marker at DNA break sites in ( c ) and ( e ). Scale bars, 10 μm. ( g ) The protein levels of repair factors in TRADD depletion. EJ-5 or DR cells were transfected with TRADD siRNAs (#1 or #2) or control siRNA. The levels of repair factors were detected by using target antibody, respectively. Total protein levels were verified with Ponceus S staining and tubulin antibody as a loading control.

Article Snippet: TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex.

Techniques: Non-Homologous End Joining, Knockdown, Transfection, Construct, Flow Cytometry, Staining, Marker, Control