neutralizing trail antibody (anti-trail  (R&D Systems)

Journal: Cancers

Article Title: Downregulation of TRAIL-Receptor 1 Increases TGFβ Type II Receptor Expression and TGFβ Signalling Via MicroRNA-370-3p in Pancreatic Cancer Cells

doi: 10.3390/cancers10110399

Figure Lengend Snippet: Knockdown of TRAIL-R1 increases the abundance of TGFβ-RII in Panc1 cells. Panc1 cells were transfected with siRNA against TRAIL-R1 or with control siRNA for 72 h without ( A ) or with ( B ) exposure to a neutralizing antibody against TRAIL (anti-TRAIL, 10 µg/mL) or ( C ) recombinant TRAIL (10 ng/mL). The expression of TRAIL-R1 and TGFβ-RII was analysed by Western blotting in whole cell lysates. As control for equal gel loading, levels of β-actin were determined in parallel. The blots shown are representative of three independent experiments yielding very similar results. ( D ) Densitometry-based quantification of the Western blots shown in ( A ). Data were compiled from three independent experiments and represent the mean ± SD ( n = 3). ( E ) Densitometry-based quantification of the Western blots shown in ( B ). ( F ) Densitometry-based quantification of the Western blots shown in ( C ). The asterisks ( * ) in ( D – F ) indicate significance relative to the ctrl.-siRNA; n.s.: not significant.

Article Snippet: Cell-stimulating agents used were TGFβ1 (ReliaTech, Wolfenbüttel, Germany), recombinant human TRAIL (10 ng/mL; PeproTech, Hamburg, Germany) and neutralizing TRAIL antibody (anti-TRAIL) (10 µg/mL; R&D Systems).

Techniques: Transfection, Recombinant, Expressing, Western Blot

Journal: Infection and Immunity

Article Title: Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1 ▿

doi: 10.1128/IAI.00342-10

Figure Lengend Snippet: Effects of Stx1, sTRAIL, and anti-TRAIL antibody on apoptosis of undifferentiated (monocytic) THP-1 cells. (A) Representative FACS scatter plots of untreated monocytic THP-1 cells and cells treated with soluble TRAIL, Stx1, or Stx1 plus TRAIL for 4 h, showing percentages of cells staining annexin V positive (lower right quadrants) or annexin V/propidium iodide (PI) double positive (upper right quadrants). (B) Monocytic THP-1 cells were either untreated or incubated with Stx1 with or without soluble TRAIL or neutralizing anti-TRAIL antibodies for 4 to 6 h. At each time point, cells were stained with annexin V and PI and analyzed by flow cytometry for apoptosis. The data shown are the means ± SEM of additive percentages of AV+ plus AV+ PI+ cells for three independent experiments. *, significant difference (P < 0.05) between cells treated with Stx1 versus Stx1 plus TRAIL for 4 h; **, significant difference (P < 0.01) between cells treated with Stx1 versus Stx1 plus anti-TRAIL antibodies for 4 h. (C) Monocytic THP-1 cells were incubated with Stx1 or Stx1A− for 4 h with or without treatment with soluble TRAIL (sTR) or neutralizing anti-sTRAIL antibodies (anti-sTR). Cells were stained with annexin V and PI, and the effects of treatment combinations on apoptosis were measured by flow cytometry. Results represent means ± SEM for three independent experiments. *, significant difference (P < 0.001) between treated cells and untreated cells; **, significant difference (P < 0.005) between cells treated with Stx1 plus sTR versus Stx1 plus anti-sTR; #, significant difference (P < 0.05) between cells treated with Stx1 versus Stx1 plus sTR; ##, significant difference (P < 0.01) between cells treated with Stx1 versus Stx1 plus anti-sTR. Cycloheximide (CHX) served as a negative control.

Article Snippet: In some experiments, neutralizing anti-tumor necrosis factor-related apoptosis-inducing ligand (anti-TRAIL) antibodies (10 to 50 ng/ml; R&D Systems, Minneapolis, MN) were added to the cells for 40 min prior to stimulation with Stx1 and incubation at 37°C in humidified 5% CO 2 .

Techniques: Staining, Incubation, Flow Cytometry, Negative Control

Journal: Infection and Immunity

Article Title: Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1 ▿

doi: 10.1128/IAI.00342-10

Figure Lengend Snippet: Effects of Stx1 and TRAIL on apoptosis in macrophage-like THP-1 cells. (A) Macrophage-like THP-1 cells were treated with Stx1, Stx1A−, sTRAIL (sTR) (50 ng/ml), or anti-TRAIL antibodies (antiTR) (50 ng/ml) for 24 h. Control, untreated cells. Cells were fixed, permeabilized, and incubated with TUNEL reaction solution. Representative TUNEL-positive cells visualized by fluorescence microscopy are shown. (B) The relative fluorescence intensity of each treatment was measured using a microplate reader. Results shown are the means ± SEM from at least three independent experiments. *, significant difference (P < 0.001) between treated cells and control cells; **, significant difference (P < 0.01) between Stx1 plus sTR and Stx1 plus anti-TR antibodies. Thapsigargin was used as a positive control for ER stress-induced apoptosis. (C) To determine the extent of cell detachment after various treatments, differentiated THP-1 cells were treated with Stx1, Stx1A−, or thapsigargin for 24 h. In some experiments, cells were pre- or posttreated with sTRAIL or anti-TRAIL antibodies. Culture supernatants were removed, and 100-μl suspensions of trypsinized THP-1 cells were transferred to microcentrifuge tubes. Trypan blue exclusion was used to distinguish viable and nonviable cells. Two hundred microliters of the trypan blue suspensions were transferred to hemocytometer chambers for counting. Viable cells were counted and expressed as a percentage of untreated control cell numbers.

Article Snippet: In some experiments, neutralizing anti-tumor necrosis factor-related apoptosis-inducing ligand (anti-TRAIL) antibodies (10 to 50 ng/ml; R&D Systems, Minneapolis, MN) were added to the cells for 40 min prior to stimulation with Stx1 and incubation at 37°C in humidified 5% CO 2 .

Techniques: Incubation, TUNEL Assay, Fluorescence, Microscopy, Positive Control

Journal: Infection and Immunity

Article Title: Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1 ▿

doi: 10.1128/IAI.00342-10

Figure Lengend Snippet: Silencing of DR5 and CHOP expression reduces CHOP-dependent expression of DR5- and Stx1-induced apoptosis in macrophage-like, but not monocytic, THP-1 cells. (A and B) Differentiated THP-1 cells were transfected with or without 200 pmol siRNA duplexes targeting DR5 (siDR5) or CHOP (siCHOP) or with nontargeting siRNA duplexes (NTsiRNA) as described in Materials and Methods. After 72 h, cells were exposed to Stx1 for 8 h, followed by Western blotting using anti-DR5 (A and B) and anti-CHOP (B) antibodies, which recognize 46-kDa (precursor form)/41-kDa (mature form) and 29-kDa proteins, respectively. β-Actin was used as an equal-protein-loading control. The bar graphs show the quantitative analysis (means ± SEM) of DR5 or CHOP protein expression from three independent experiments. *, significant difference in DR5 protein expression (P < 0.01; Stx1 versus siRNA DR5 plus Stx1 treatment). (C) Effect of DR5 or CHOP knockdown on apoptosis of macrophage-like THP-1 cells. Differentiated THP-1 cells were transfected with DR5 siRNA (siDR5), CHOP siRNA (siCHOP), or nontargeting siRNA (NTsiRNA) or without siRNA (mock) for 72 h, followed by Stx1 treatment for another 24 h. Cells were fixed, permeabilized, and incubated with TUNEL reaction solution or DAPI to stain nuclei. Cells were visualized by fluorescence microscopy, and 10 different fields (magnification, ×20) for each treatment were selected to count total numbers of TUNEL positive cells. Total numbers of cells per field (magnification, ×20) were also counted, and the percentage of apoptotic cells above that for untreated controls were calculated as described in Materials and Methods. Upper panels, representative fields showing TUNEL-positive cells from each treatment. Lower bar graph, quantitative data showing percentage of apoptosis (means ± SEM from at least four independent experiments) for each treatment. *, significant difference (P < 0.001) for cells treated with Stx1 alone versus siDR5- plus Stx1-treated cells. (D) Monocytic THP-1 cells transfected with siRNAs specific for DR5 (siDR5), CHOP (siCHOP), or nontargeting siRNA (NTsiRNA) or mock transfected were treated with Stx1 for 5 h. Expression of DR5 and CHOP was assessed by Western blotting as outlined above. The bar graph depicts quantitative analysis (means ± SEM) of DR5 or CHOP protein expression from three independent experiments. *, significant difference in DR5 and CHOP protein expression (P < 0.01). (E) Quantitative data showing percentages of apoptosis for each treatment from the FACS scatter plots of untreated monocytic THP-1 cells, cells treated with Stx1 alone, or cells treated with siRNAs plus Stx1. Results are the means ± SEM from at least four independent experiments.

Article Snippet: In some experiments, neutralizing anti-tumor necrosis factor-related apoptosis-inducing ligand (anti-TRAIL) antibodies (10 to 50 ng/ml; R&D Systems, Minneapolis, MN) were added to the cells for 40 min prior to stimulation with Stx1 and incubation at 37°C in humidified 5% CO 2 .

Techniques: Expressing, Transfection, Western Blot, Incubation, TUNEL Assay, Staining, Fluorescence, Microscopy

Journal: Infection and Immunity

Article Title: Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1 ▿

doi: 10.1128/IAI.00342-10

Figure Lengend Snippet: CHOP and DR5 signaling activates the mitochondrion-mediated pathway of apoptosis in Stx1-treated macrophage-like THP-1 cells. Differentiated THP-1 cells were transfected with siRNAs specific for DR5 (siDR5), CHOP (siCHOP), or nontargeting siRNA (NTsiRNA) or were mock transfected (Mock) with reagent only for 72 h and then treated without or with Stx1, Stx1A−, or Stx1 B subunits for 24 h. After incubation, JC-1 was used to measure ΔΨm (loss of mitochondrial membrane potential) by flow cytometry. Control cells were maintained in medium for 24 h. The percent ΔΨm was compared to that for control (untreated) cells. The data are the means ± SEM from three independent experiments.

Article Snippet: In some experiments, neutralizing anti-tumor necrosis factor-related apoptosis-inducing ligand (anti-TRAIL) antibodies (10 to 50 ng/ml; R&D Systems, Minneapolis, MN) were added to the cells for 40 min prior to stimulation with Stx1 and incubation at 37°C in humidified 5% CO 2 .

Techniques: Transfection, Incubation, Membrane, Flow Cytometry