tradd plasmid  (OriGene)

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy

doi: 10.1152/ajpendo.00215.2015

Figure Lengend Snippet: TRADD in vitro overexpression induces unfolded protein response (UPR) and ER stress. A: TRADD mRNA and protein levels after TRADD plasmid transfections. Control (Ctrl) vector: the backbone vector of the TRADD construct. B: protein levels of p-IRE1α, p-PERK, p-eIF2α, BiP, and CHOP after TRADD plasmid transfections; Ctrl vector transfection. C: protein levels of p-ASK1 and p-JNK1/2 after TRADD plasmid transfections. D: miRNA levels. E: representative images of the TUNEL assay. Apoptotic cells were labeled as red, and cell nuclei were stained by DAPI (blue). F: levels of cleaved caspase 3; experiments were repeated 3 times (n = 3). G: levels of cleaved caspase 8; experiments were repeated 3 times (n = 3). *Significant differences compared with other groups (in A, B, C, D, F, and G); * and #significant differences (P < 0.05) compared with each other or between the other two groups (in E).

Article Snippet: TRADD plasmid (Myc-DDK-tagged, MR223646 ) was obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: In Vitro, Over Expression, Plasmid Preparation, Transfection, Control, Construct, TUNEL Assay, Labeling, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma 1 2

doi:

Figure Lengend Snippet: (A) U251MG cells were infected with control (scrambled) lentivirus or with TRADD shRNA lentivirus, followed by puromycin selection and Western blot of clones infected with TRADD shRNA. Clones 15 and 26 show the best silencing of TRADD. (B) Degradation of IκBα in response to TNF-α (20 ng/ml) can be seen in U251MG control shRNA cells, showing that TNF-α activates NF-κB in these cells. (C and D) Failure of IκBα degradation in response to TNF-α in TRADD-silenced clones 15 and 26, demonstrating that TRADD is essential for NF-κB activation. (E) A reporter assay in clone 26 with an NF-κB responsive luciferase reporter. While NF-κB transcriptional activity increases in response to TNF-α in control shRNA cells, there is no increase in TRADD-silenced clone 26, again confirming the requirement for TRADD in NF-κB activation (one-way ANOVA, P = .0031). (F) Western blot showing that phosphorylation of the p65 subunit of NF-κB is in response to TNF-α is impaired in TRADD-silenced clone 26. (G) Quantitative real-time polymerase chain reaction experiment showing that mRNA levels of the NF-κB target gene IL-8 is decreased in TRADD-silenced clone 26, both at a basal level (P ≤ .0001) and in response to TNF-α (P ≤ .0001) compared to control. Newman-Keuls multiple comparison test was used for statistical analysis.

Article Snippet: RNA Interference TRADD was silenced using lentiviral shRNA ready-to-use transduction particles from Santa Cruz Biotechnology (sc-36709-V, containing three target-specific constructs) and puromycin selection according to the manufacturer's protocol.

Techniques: Infection, Control, shRNA, Selection, Western Blot, Clone Assay, Activation Assay, Reporter Assay, Luciferase, Activity Assay, Phospho-proteomics, Real-time Polymerase Chain Reaction, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma 1 2

doi:

Figure Lengend Snippet: (A) An MTT conversion assay was conducted comparing U251 cells with control shRNA or stably silenced TRADD (clones 15 and 26). MTT conversion is higher in control cells compared to TRADD-silenced cells suggesting increased proliferation in control cells (one-way ANOVA, P = .005). Cell death was excluded by trypan blue exclusion in parallel experiments. (B) Analysis of cell proliferation in U251 TRADD-silenced and control shRNA clones. 5K cells were plated onto 24-well dishes for 72 hours followed by trypsinization and counting in an automated cell counter. Cell proliferation is decreased in TRADD-silenced clones 15 and 26 (one-way ANOVA, P = .04). (C) An Annexin-FACS assay was conducted to determine if TRADD silencing leads to cell death. The percentage of viable cells was compared in control and TRADD-silenced cells by unpaired t test. No cell death was detected in TRADD-silenced clone 26. (D) An MTT conversion assay was conducted in U251 cells with control shRNA or TRADD-silenced clone 26 to assess the response to increasing doses of temozolomide. While U251 control shRNA cells are somewhat resistant to temozolomide, silencing TRADD increases the vulnerability of cells to temozolomide at 10 µM (P ≤ .04), 25 µM (P ≤ .04), and 50 µM (P ≤ .0003) in comparison to the control shRNA cells. Data were analyzed by two-tailed unpaired t test.

Article Snippet: RNA Interference TRADD was silenced using lentiviral shRNA ready-to-use transduction particles from Santa Cruz Biotechnology (sc-36709-V, containing three target-specific constructs) and puromycin selection according to the manufacturer's protocol.

Techniques: Control, shRNA, Stable Transfection, Clone Assay, Comparison, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma 1 2

doi:

Figure Lengend Snippet: (A) A panel of glioma stem-like cultures grown as neurospheres from various GBMs was screened for the presence of TRADD by Western blot. All of the stem cell cultures express TRADD. (B) TRADD was silenced in neurosphere culture derived from GBM836 using lentiviral shRNA followed by puromycin selection, and two pools were examined. Pool 2 has better silencing of TRADD. Control shRNA (scrambled) cells were used as control. (C) While the majority of control shRNA cells are viable, silencing TRADD in glioma stemcell cultures results in cell death with increased Annexin-positive cells (P ≤ .008, two-tailed unpaired t test). (D) A second GBM primary neurosphere culture from GBM622 with silenced TRADD also shows increased Annexin-positive cells in TRADD-silenced cells compared to control shRNA cells (P ≤ .0006, two-tailed unpaired t test). (E) Western blot showing silencing of TRADD in GBM622 neurosphere cultures.

Article Snippet: RNA Interference TRADD was silenced using lentiviral shRNA ready-to-use transduction particles from Santa Cruz Biotechnology (sc-36709-V, containing three target-specific constructs) and puromycin selection according to the manufacturer's protocol.

Techniques: Western Blot, Derivative Assay, shRNA, Selection, Control, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma 1 2

doi:

Figure Lengend Snippet: Impaired NF-κB activation in TRADD-silenced glioma stem cell cultures (836, pool 2). (A) Phosphorylation of the p65 subunit of NF-κB decreased in TRADD-silenced glioma stem cell cultures compared to control shRNA cells. (B) A reporter assay in TRADD-silenced cells with an NF-κB responsive luciferase reporter. NF-κB transcriptional activity is significantly higher in control cells compared to TRADD-silenced cells. Cells were treated with TNF-α for 6 hours. (C) Quantitative real-time polymerase chain reaction experiment showing that mRNA levels of the NF-κB target gene IL-8 - increases after 6 hours of TNF-α exposure in control cells, while it does not increase in TRADD-silenced cells (P ≤ .0001). TRADD silencing is shown in Figure 6B. Newman-Keuls multiple comparison test was used for statistical analysis.

Article Snippet: RNA Interference TRADD was silenced using lentiviral shRNA ready-to-use transduction particles from Santa Cruz Biotechnology (sc-36709-V, containing three target-specific constructs) and puromycin selection according to the manufacturer's protocol.

Techniques: Activation Assay, Phospho-proteomics, Control, shRNA, Reporter Assay, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Comparison