trail r2 fc  (ATCC)

Journal: Nature Nanotechnology

Article Title: A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns

doi: 10.1038/s41565-024-01676-4

Figure Lengend Snippet: a , Experimental setup of cancer cell treatment with Cy5-labelled origami at pH 7.4 or 6.5. b , The Cy5 signal of SK-BR-3 cells treated with 5 nM empty origami, PEG_pH_O 6p at pH 7.4 or 6.5, measured by flow cytometry. c , Localization of Cy5-labelled origamis (magenta) relative to DR5 (cyan; detected using Alexa488-labelled anti-DR5 monoclonal antibody) on SK-BR-3 cells. The co-localization of the magenta and cyan is shown in grey. Maximum intensity projections from z -stacks. Scale bars, 20 μm.

Article Snippet: The DR5 analyte (R&D Systems, 10140-T2) was diluted from 516 nM to 32.25 nM and injected as a single-cycle kinetics experiment, with a flow rate of 30 µl min −1 , and 500 s contact time for each concentration.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

doi: 10.1101/2023.07.26.550758

Figure Lengend Snippet: (A) The sensitivity of U266 and IM-9 cells to TRAIL (10, 30, 100, 300ng/ml) was determined by Annexin V-FITC assay. (B) TRAIL (100 ng/ml)-induced apoptosis of IM-9 and U266 cells, with or without HL-III pretreatment, were determined by Annexin V-FITC assay. (C) Expression of TRAIL receptor DR4 and DR5 on U266B1, RPMI-8226 and IM-9 cells were determined with PE conjugated mAbs against DR4 and DR5 using FACS. The shaded histograms are from cells stained with mouse IgG1-PE conjugate. (D) Expressions of cell surface HS on untreated cells or cells pretreated with HL-III were determined by a human anti-HS mAb, followed by an anti-human-IgG Alexa-594 secondary antibody. The shaded histogram is from cells stained with secondary antibody only. (E) Expression of cell surface syndecan-1 was determined by a mouse anti- syndecan-1 mAb followed by an anti-mouse-IgG Alexa-488 secondary antibody. The shaded histogram is from cells stained with secondary antibody only. Error bars represent S.D. ** represents p < 0.01 and *** represents p < 0.0001 by Student’s t test. Data are representative of at least three separate assays.

Article Snippet: Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10µg), or DR5– mTRAIL complex (10 µg each pre-incubated for 1 h at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 µl bed volume).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

doi: 10.1101/2023.07.26.550758

Figure Lengend Snippet: (A) While DR5 does not bind heparin by itself (left half of the gel), DR5-TRAIL complex can bind heparin (right half of the gel), indicating DR5-TRAIL-heparin can form a ternary complex through TRAIL. Representative of three experiments with identical results (B) DR5 and HS bind to different surfaces on TRAIL. Crystal structure of hTRAIL-DR5 complex (IDU3). hTRAIL is shown in cartoon and the three monomers are displayed in green, salmon and gold, respectively. The three DR5 molecules are shown in gray cartoon. Because residues 114- 119 of hTRAIL are disordered in this structure, these residues ( 114 VRERGP 119 , backbone shown in gray random coils) were manually modeled onto the last visible N-terminal residue (Q120) of the hTRAIL. Sidechains responsible for HS binding (from R115, R117 and R121) are shown in sticks. (C-E) TRAIL-dependent internalization of DR4 and DR5 was determined by a FACS-based assay. Cell surface levels of TRAIL receptor DR5(C) and DR4 (D) were determined before TRAIL stimulation, and 30min and 1h after TRAIL stimulation. The shaded histograms are from cells stained mouse IgG1-PE conjugate. (E) Plot of time-dependent internalization of cell surface DR5, with or without HL-III treatment. n = 3. Data is representative of three experiments with similar results.

Article Snippet: Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10µg), or DR5– mTRAIL complex (10 µg each pre-incubated for 1 h at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 µl bed volume).

Techniques: Binding Assay, Staining

Journal: PLoS ONE

Article Title: Astrocytic α V β 3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

doi: 10.1371/journal.pone.0034295

Figure Lengend Snippet: ( A–D ) Quantification of four different morphological parameters using IMARIS software (Bitplane, Switzerland) of bright-field microscopy images of CAD cells seeded over plastic, seeded over plastic in serum-free medium previously conditioned by DITNC1 cells for 24 hours (black bars) or over a monolayer of DITNC1 astrocytes in serum-free medium. ( A ) Percentage of differentiated CAD cells with processes ≥15 µm; ( B ) length of the processes extended by differentiated cells, expressed as a percentage of the control over plastic; ( C ) number of processes in 100 differentiated cells and ( D ) number of varicosities per 100 differentiated cells. ( E,F ) CAD cells seeded over fixed-astrocyte monolayers were induced to differentiate. To block α V β 3 integrin, fixed-cells were incubated with recombinant Thy-1-Fc or antibodies against β 3 integrin. TRAIL-R2-Fc or antibodies against β 1 integrin were used as controls. Co-cultures were photographed ( E ) and the percentage of differentiated CAD cells ( F ) was quantified as in ( A ). Arrows in E indicate axon-like neurites growing over the DITNC1 mololayer. All graphs show mean+s.e.m. determined from at least 100 cells per condition; n = 3. ** P <0.01 or * P <0.05 compared with control cells seeded over plastic. # P <0.05 compared with their respective control.

Article Snippet: Then, fixed monolayers were incubated with Thy-1-Fc or TRAIL-R2-Fc recombinant proteins (600 ng/ml) or with anti-β 3 integrin (clone F11, Pharmingen) or anti-β 1 integrin (clone Ha2/5, Beckton & Dickinson) antibodies (5 µg/ml) during 1 hour.

Techniques: Software, Microscopy, Blocking Assay, Incubation, Recombinant

Journal: PLoS ONE

Article Title: Astrocytic α V β 3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

doi: 10.1371/journal.pone.0034295

Figure Lengend Snippet: ( A ) Cortical neurons, cultured for 4 and 7 days of culture in vitro were stained for Thy-1 (red), MAP-2 (soma/dendrite staining, blue) and Tau (axon staining, green). Arrows indicate some areas with Thy-1 staining. Asterisks label Thy-1-negative axons. ( B–E ) Neurons, cultured for 4–5 days in vitro were treated with supernatants containing α V β 3 -Fc fusion protein ( α V β 3 -Fc ), α V β 3 -Fc-depleted supernatants ( DS ), α V β 3 -Fc-depleted supernatants supplemented with TRAIL-R2-Fc ( TRAIL-R2-Fc ), or non-treated ( NT ) for 72 hours. Where indicated, 1 U/ml of PI-PLC was added prior to integrin addition ( D,E ). ( B,D ) Inverted fields of MAP-2 fluorescence images that were used to count neurons and evaluate dendrite length. ( C,E ) Quantification of two different morphological parameters performed using Neuro ImageJ software. shown are the mean+s.e.m. of 720 neurons from three independent experiments ( C ) or 240 neurons from two independent experiments ( E ). * P <0.05 compared with DS condition.

Article Snippet: Then, fixed monolayers were incubated with Thy-1-Fc or TRAIL-R2-Fc recombinant proteins (600 ng/ml) or with anti-β 3 integrin (clone F11, Pharmingen) or anti-β 1 integrin (clone Ha2/5, Beckton & Dickinson) antibodies (5 µg/ml) during 1 hour.

Techniques: Cell Culture, In Vitro, Staining, Fluorescence, Software

Journal: Molecular Cancer

Article Title: 2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

doi: 10.1186/1476-4598-8-122

Figure Lengend Snippet: 2-DG up-regulates TRAIL death receptors in melanoma cells . A , Mel-RM and MM200 cells treated with 2-DG (10 μM) for indicated periods were subjected to measurement of the cell surface expression of TRAIL-R2 (upper panel) and -R1 (lower panel) using flow cytometry. The data in y axes represent mean fluorescence intensity (MFI). B , Representative flow cytometry histograms showing up-regulation of TRAIL-R2 by 2-DG in melanoma cells. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. C , 2-DG up-regulates TRAIL-R2 and -R1 in a panel of melanoma cell lines, melanocytes, and fibroblasts. Cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 (upper panel), and -R1 (lower panel) was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D , Whole cell lysates from Mel-RM and MM200 cells treated with 2-DG (10 μM) for indicated periods were subjected to Western blot analysis. E , Left panel: Mel-RM and MM200 cells were treated with the 2-DG (10 μM) for indicated periods. Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. The increases in TRAIL-R2 mRNA at 16, 24, and 36 hours after treatment in both cell lines were statistically significant (p < 0.05); Right panel: Mel-RM and MM200 cells were treated with actinomycin D (3 μg/ml) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Up-regulation of TRAIL-R2 mRNA by 2-DG was significantly inhibited by actinomycin D in both cell lines (p < 0.05). The data shown are either the mean ± SE (A, C, & E), or representative (B & D), of three individual experiments.

Article Snippet: Recombinant human TRAIL and the TRAIL-R2/Fc chimera were supplied by Genentech Inc. (San Francisco, CA).

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: 2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

doi: 10.1186/1476-4598-8-122

Figure Lengend Snippet: Sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG is largely due to up-regulation of TRAIL-R2 . A , Mel-RM and MM200 cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. B , Mel-RM and M200 cells were treated the caspase-8 specific inhibitor z-IETD-fmk (30 μM) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. C , Mel-RM and MM200 cells were treated with a TRAIL-R1/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. D , Mel-RM and MM200 cells were transfected with the control or TRAIL-R2 siRNA. Left panel: Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. Right panel: Twenty-four hours later, the cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, C, & the right panel of D), or representative (the left panel of D), of three individual experiments.

Article Snippet: Recombinant human TRAIL and the TRAIL-R2/Fc chimera were supplied by Genentech Inc. (San Francisco, CA).

Techniques: Flow Cytometry, Transfection, Western Blot

Journal: Molecular Cancer

Article Title: 2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

doi: 10.1186/1476-4598-8-122

Figure Lengend Snippet: 2-DG-mediated up-regulation of TRAIL-R2 is independent of p53 and CHOP . A , Mel-RM and MM200 cells were transfected with the control or p53 siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. B , Mel-RM and MM200 cells were transfected with the control or p53 siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for a further 16 hours. Upper panel: The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in y axes represent mean fluorescence intensity (MFI). Lower panel: Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. C , Whole cell lysates from Mel-RM and MM200 cells transduced with the control or CHOP shRNA were subjected to Western blot analysis. The arrow head points to non-specific bands generated by the antibody against CHOP. Note that the cells were treated with TM (3 μM) for 16 hours before harvest to better visualize CHOP. D , Mel-RM and MM200 cells transduced with the control or CHOP shRNA were treated with 2-DG (10 μM) for 16 hours. Left panel: The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). Right panel: Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. The data shown are either the mean ± SE (B & D), or representative (A & C), of three individual experiments.

Article Snippet: Recombinant human TRAIL and the TRAIL-R2/Fc chimera were supplied by Genentech Inc. (San Francisco, CA).

Techniques: Transfection, Western Blot, Expressing, Flow Cytometry, Fluorescence, Isolation, Real-time Polymerase Chain Reaction, Transduction, shRNA, Generated

Journal: Molecular Cancer

Article Title: 2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

doi: 10.1186/1476-4598-8-122

Figure Lengend Snippet: XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG . A , 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B , Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C , Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D , Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E , Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.

Article Snippet: Recombinant human TRAIL and the TRAIL-R2/Fc chimera were supplied by Genentech Inc. (San Francisco, CA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Derivative Assay, Transfection, Expressing, Flow Cytometry, Fluorescence, Transduction, shRNA, Isolation, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: 2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

doi: 10.1186/1476-4598-8-122

Figure Lengend Snippet: 2-DG up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in fresh melanoma isolates . A , Mel-CA and Mel-MC with (thick open histograms) or without (thin open histograms) treatment with 2-DG (10 μM) for 16 hours were subjected to measurement of the cell surface TRAIL-R2 expression in flow cytomety. The filled histograms are isotype controls. B , Whole cell lysates from Mel-CA and Mel-MC with or without treatment with 2-DG (10 μM) for 16 hours were subjected to Western blot analysis. C , Freshly isolated melanoma cells were treated with 2-DG (10 μM), TRAIL (200 ng/ml), or the combination of both for 24 hours were subjected to measurement of apoptosis by the propidium iodide method using flow cytometry. D , Freshly isolated melanoma cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (C & D), or representative (A & B), of three individual experiments.

Article Snippet: Recombinant human TRAIL and the TRAIL-R2/Fc chimera were supplied by Genentech Inc. (San Francisco, CA).

Techniques: Expressing, Western Blot, Isolation, Flow Cytometry