Journal: Cancers

Article Title: Targeting of CD133+ Cancer Stem Cells by Mesenchymal Stem Cell Expressing TRAIL Reveals a Prospective Role of Apoptotic Gene Regulation in Non-Small Cell Lung Cancer

doi: 10.3390/cancers11091261

Figure Lengend Snippet: TRAIL sensitivity by in vitro characterization. ( A ) The half maximal inhibitory concentration (IC 50 ) value was determined in non-small cell lung cancer (NSCLC) cell lines (H460, H2170 and A549) by MTS assay after 48 h of rhTRAIL treatment at different concentrations. ( B ) Analysis of TRAIL receptors expression [agonist receptors (DR4 and DR5) and decoy receptors (DcR1 and DcR2)] in MSCs and NSCLC cell lines by flow cytometry. ( C ) Sensitivity of NSCLC cell lines and MSCs evaluated by MTS assay after rhTRAIL (500 ng/mL) treatment.

Article Snippet: The NSCLC cell lines were analyzed for TRAIL agonist (DR4 and DR5) and other decoy receptors (DcR1 and DcR2) with allophycocyanin (APC)-conjugated TRAIL-R1 (DR4) (cat no: FAB347A; Mouse IgG1), TRAIL-R2 (DR5) (cat no: FAB6311A; Mouse IgG 2B ), TRAIL-R3 (DcR1) (cat no: FAB6302A; Mouse IgG1), and TRAIL-R4 (DcR2) (cat no: FAB633A; Mouse IgG1) (R&D Systems, Minneapolis, MN, USA) using flow cytometry (Becton Dickinson BD).

Techniques: In Vitro, Concentration Assay, MTS Assay, Expressing, Flow Cytometry

Journal: Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases

Article Title: Identifying patients with bacterial infections using a combination of C-reactive protein, procalcitonin, TRAIL, and IP-10 in the emergency department: a prospective observational cohort study.

doi: 10.1016/j.cmi.2018.09.007

Figure Lengend Snippet: Fig. 2. ROC curves of CRP, PCT, TRAIL and IP10 for suspected and confirmed bacterial infections. AUC, area under curve; CRP, C-reactive protein; IP-10, interferon-gamma induced protein-10; PCT, procalcitonin; ROC curve, receiver operator characteristic curve; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

Article Snippet: TRAIL and IP-10 levels were determined using Human TRAIL/ TNFSF10 Quantikine ELISA Kit and Human CXCL10/IP-10 Quantikine ELISA Kit of R&D Systems according to the manufacturer's manual.

Techniques:

Journal: Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases

Article Title: Identifying patients with bacterial infections using a combination of C-reactive protein, procalcitonin, TRAIL, and IP-10 in the emergency department: a prospective observational cohort study.

doi: 10.1016/j.cmi.2018.09.007

Figure Lengend Snippet: Fig. 3. ROC curves of CRP, PCT, TRAIL and IP10 for suspected and confirmed bacterial infections. AUC, area under curve; CRP, C-reactive protein; IP-10, interferon-gamma induced protein-10; PCT, procalcitonin; ROC curve, receiver operator characteristic curve; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

Article Snippet: TRAIL and IP-10 levels were determined using Human TRAIL/ TNFSF10 Quantikine ELISA Kit and Human CXCL10/IP-10 Quantikine ELISA Kit of R&D Systems according to the manufacturer's manual.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Proteasome inhibitor PS-341 (VELCADE) induces stabilization of the TRAIL receptor DR5 mRNA through the 3′-untranslated region

doi: 10.1158/1535-7163.mct-07-2368

Figure Lengend Snippet: Figure 3. PS-341 treatment increases the binding of proteins to the fragments of the 3¶-UTR. A, schematic representation of the DR5 mRNA and the fragments of 3¶-UTR. B, autoradiographic images of the RNA-protein interactions shown by UV cross-linking experiments. Fifty micrograms of cytoplasmic extracts from LNCaP cells treated with or without PS-341 (500 nmol/L, 18 h) were incubated with in vitro transcribed, radiolabeled F-1, F-2, F-3, F-4, and F-5 transcripts and UV cross-linked for 30 min. Excess RNA was digested with RNase cocktail and the cross-linked proteins were resolved via 10% SDS- PAGE. The gel was dried and subjected to autoradiography. C, time course of binding of PS-341-induced proteins to the F-1 fragment. Experiment was carried out as in B using cytoplasmic extracts from LNCaP cells at varying times after PS-341 treatment. D, stabilization of EGFP mRNA by the 3¶-UTR fragments F-1 and F-4. The experiment was carried out identically to Fig. 2, except that only a portion of the 3¶-UTR of DR5 was cloned after the EGFP reporter.

Article Snippet: Plasmid Constructs andTransfection A full-length DR5 clone was obtained from Origene.

Techniques: Binding Assay, Incubation, In Vitro, SDS Page, Autoradiography, Clone Assay

Journal: Molecular Cancer Therapeutics

Article Title: Proteasome inhibitor PS-341 (VELCADE) induces stabilization of the TRAIL receptor DR5 mRNA through the 3′-untranslated region

doi: 10.1158/1535-7163.mct-07-2368

Figure Lengend Snippet: Figure 5. Analysis of RNA-binding proteins in LNCaP cells treated with PS-341. A, Western blot analysis of cytoplasmic (Cyto) and nuclear extracts (Nuc) prepared from vehicle or PS-341-treated (0.5 Amol/L, 18 h) LNCaP cells. Cytoplasmic extracts (30 Ag) and nuclear extracts (10 Ag) were run on 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies to specific RNA-binding proteins. GAPDH was used as a loading control for the cytoplasmic extract, and Lamin B1 was a control for equivalent loading of nuclear proteins. B, PCR analysis of endogenous DR5 mRNA after immunoprecipitation of mRNA-binding proteins. Cytoplasmic extracts prepared from vehicle or PS-341-treated (500 nmol/L, 18 h) LNCaP cells were incubated with monoclonal HuR antibody, monoclonal TIAR antibody, or nonspecific IgG-coated protein A beads and immunoprecipitated in RNase- free conditions. RNA extracted from the immunoprecipitated complex was subjected to reverse transcription and PCR for 25 cycles. Bottom, bar graph of the relative RNA levels. Experiments were repeated in triplicate and the mean F SD is plotted. To establish equivalent levels of RNA in the samples, total RNA extracted from the cytoplasmic extracts was run on 1% formaldehyde agarose gel and ribosomal bands were photographed (see inset, lane 1 for vehicle and lane 2 for PS-341 treated). C, quantitative real-time PCR analysis of endogenous DR5 mRNA immunoprecipitated with various mRNA-binding proteins. Immunoprecipitation was carried out on extracts generated as described in B. The RNA extracted from the immunoprecipitated complex was reverse transcribed and subjected to quantitative real-time PCR analysis. The cytoplasmic extracts (input) are shown. Columns, fold increase in DR5 mRNA immunoprecipitated. The experiments were repeated in triplicate and the mean F SD is plotted. D, autoradiographic images of experiments carried out in a similar fashion to B using in vitro transcribed and radiolabeled F-1 and F-12 transcripts. LNCaP cells were treated with DMSO or PS-341 (500 nmol/L, 18 h) and UV cross-linked with F-1 and F-12 transcripts. RNA-binding proteins were immunoprecipitated with HuR or TIAR antibodies or nonspecific IgG. After immunoprecipitation, the complexes were separated via 10% SDS-PAGE and subjected to phosphorimaging. One tenth of the samples used for immunoprecipitation was loaded in lanes 1 and 9 without immunoprecipitation. Bottom, quantitative representation of the radioactivity in the transcripts immunoprecipitated with HuR antibody. The experiments were repeated in triplicate and the mean F SD is plotted.

Article Snippet: Plasmid Constructs andTransfection A full-length DR5 clone was obtained from Origene.

Techniques: RNA Binding Assay, Western Blot, SDS Page, Membrane, Control, Immunoprecipitation, Binding Assay, Incubation, Reverse Transcription, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Generated, In Vitro, Radioactivity

Journal: Molecular Cancer Therapeutics

Article Title: Proteasome inhibitor PS-341 (VELCADE) induces stabilization of the TRAIL receptor DR5 mRNA through the 3′-untranslated region

doi: 10.1158/1535-7163.mct-07-2368

Figure Lengend Snippet: Figure 6. HuR stabilizes DR5 mRNA in LNCaP cells. A, Western blot analysis of samples from LNCaP cells transfected with scrambled or HuR siRNA. LNCaP cells (1 107) were electroporated with 150 pmol of either scrambled or HuR siRNA. Twenty-four hours after transfection, the cells were treated with PS-341 (500 nmol/L, 16 h) and the harvested cytoplasmic proteins were subjected to Western blot analysis. The blots were probed with HuR or GAPDH antibodies as indicated. B, autoradiographic images of the UV cross-linking experiments done with radioactive F-122 and F-421 transcripts and cytoplasmic extracts prepared from LNCaP cells transfected with scrambled, HuR, or AUF-1 siRNA duplexes. Twenty-four hours after transfection, cells were treated with PS-341 (500 nmol/L, 16 h), and the harvested cytoplasmic extracts were used for the analysis. UV cross-linking experiments were done as indicated in Materials and Methods. Arrows, molecular weights of protein standards and the HuR protein. C, quantitative real-time PCR analysis of RNA samples isolated from LNCaP cells transfected with scrambled or HuR siRNA. After 24 h of transfection, cells were treated with DMSO or PS-341 (100 nmol/L, 12 h) followed by actinomycin D (2 Ag/mL), and RNA was harvested at the times indicated. DR5 mRNA levels were normalized with endogenous GAPDH mRNA. Experiments were repeated in triplicate and the mean F the SD is plotted with the half-life of the mRNA (solid horizontal line). Closed circles, results from the scrambled siRNA; open circles, results from the cells treated with the siRNA for HuR. D, Western blot analysis was done for LNCaP cells transfected with Flag-HuR alone or along with a HA-ubiquitin cDNA construct. Twenty-four hours after transfection, cells were treated with PS-341 (500 nmol/L, 16 h) and the total extracts were prepared. The extracts were either loaded directly on SDS-PAGE or subjected to Flag (lanes 5-10) or HA (lanes 11-14) immunoprecipitation followed by either Flag (lanes 1-8 and 11-14) or HA (lanes 9 and 10) antibody Western blotting as indicated. One tenth of the total protein used for immunoprecipitation was loaded in lanes 1 to 4. Right, brackets, higher molecular weight bands consistent with multiubiquitinated HuR; left, blunt arrows, expression of Flag-HuR and its fragments.

Article Snippet: Plasmid Constructs andTransfection A full-length DR5 clone was obtained from Origene.

Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction, Isolation, Ubiquitin Proteomics, Construct, SDS Page, Immunoprecipitation, Molecular Weight, Expressing

Journal: Arthritis and rheumatism

Article Title: Mechanisms and clinical relevance of TRAIL-triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis.

doi: 10.1002/art.30181

Figure Lengend Snippet: Figure 4. Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) produce low levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-8 when stimulated with TRAIL. RA FLS (n 5) were treated with TRAIL (1 nM) or tumor necrosis factor (TNF; 0.5 nM) for 72 hours, and supernatants were analyzed for IL-6 (A), IL-8 (B), and RANTES (C) secretion by enzyme-linked immunosorbent assay. Cy- tokine levels were analyzed using Wilcoxon’s test. Values are the mean SEM. NS not stimulated.

Article Snippet: Serum levels of TRAIL and OPG were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (Quantikine human OPG ELISA Kit [R&D Systems] and a human TRAIL ELISA [Diaclone]).

Techniques: Enzyme-linked Immunosorbent Assay