Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Primary human CD4 T cells from eight different donors ( n = 8) were separately infected with lab‐adapted and primary HIV‐1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry 4 days after infection. Uninfected CD4 T cells were determined as CD4‐positive and HIV‐1 p24‐negative, infected cells as CD4‐negative and p24‐positive. Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with NL4‐3, JR‐CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity ( x ‐axis). Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles), and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4‐3: n = 8; JR‐CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7–8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary AB‐only control ( y ‐axis). Data information: Wilcoxon signed‐rank test. Values for non‐infected and infected cells of the same donor are connected with lines. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Infection, Expressing, Flow Cytometry, Incubation, Fluorescence, Comparison, Control

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Degranulation of primary human NK cells after co‐culture with various target cells in the presence or absence of αTRAIL or αDR4/5. Comparison of CD107a expression after co‐culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry ( n = 10 different donors per condition). Each data point represents the mean of two technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation ( y ‐axis) after co‐culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 10 different donors per condition). Comparison of CD107a expression after co‐culture with autologous HIV‐I‐infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry ( n = 9 different donors per condition). Each data point represents the mean of two technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation ( y ‐axis) after co‐culture with autologous HIV‐I‐infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 9 different donors per condition). Representative histograms (overlay, left panel) and bar graphs ( n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of three technical replicates. Comparison of CD107a expression after co‐culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each) or 20 µg/ml isotype control (Iso) using flow cytometry ( n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation after co‐culture with 721.221 target cells in the presence of either isotype or αDR4/5 as relative reduction compared to no antibody ( n = 9 different donors per condition). Data information: Wilcoxon signed‐rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Co-Culture Assay, Comparison, Expressing, Control, Flow Cytometry, Fluorescence, Inhibition, Infection

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Degranulation of primary human NK cells after incubation with plate‐coated antibodies or whole proteins. Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry ( n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity ( y ‐axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated antibodies of different concentrations ( x ‐axis). Comparison of CD107a expression after incubation with plate‐coated DR4 protein, DR5 protein, or human IgG using flow cytometry ( n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity ( y ‐axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated proteins of different concentrations ( x ‐axis). Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Correlation analysis between relative frequency of CD107a + NK cells and granzyme B concentration ( n = 53, data points obtained from A, B, and C, 11 different donors). Comparison of CD107a expression after incubation with plate‐coated DcR1 protein, osteoprotegerin (OPG), or human IgG using flow cytometry ( n = 9 different donors). Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated proteins of different concentrations ( x ‐axis). Data information: Wilcoxon signed‐rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, C, E) Each data point represents the mean of two technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Incubation, Comparison, Expressing, Flow Cytometry, Fluorescence, Concentration Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Lysis of different target cells in co‐culture with NK cells was quantified in various cytotoxicity assays. Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining ( y ‐axis) after co‐culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference in target cells remaining ( y ‐axis) between αTRAIL and isotype conditions displayed as p.p. ( n = 12 different donors). Each data point represents the mean of at least two technical replicates. Left panel: Representative contour plots showing the percentage of .221‐DR4/5KO (control) and .221‐Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221‐DR4/5KO and .221‐Cas9 cells ( y ‐axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221‐Cas9 cells displayed as percent ( n = 12 different donors). Each data point represents the mean of at least three technical replicates. Left panel: Representative contour plots showing the percentage of Raji‐pSIP (control) and Raji‐DR5 ++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji‐pSIP and Raji‐DR5 ++ ( y ‐axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji‐DR5 ++ cells displayed as % ( n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed‐rank test. Experiments were performed in four batches with three different donors each. “No NK” control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated “No NK” control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Lysis, Co-Culture Assay, Control

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: The expression of DR4 and DR5 was assessed by flow cytometry. 721.221 and Raji cells were labeled with LIVE/DEAD Fixable Near‐IR Stain, followed by incubation with biotin‐conjugated mouse anti‐human DR4 or DR5, and then labeled with Streptavidin‐BV421. Expression was quantified as fluorescence intensity. Representative histogram of DR4 (light orange) and DR5 (dark orange) expression in comparison to the Streptavidin‐only control (grey) or the FMO control (dashed line). Upper panel (from left to right): untransduced 721.221 cells, Cas9‐transduced .221s, and DR4/5 double knockout .221s. Lower panel (from left to right): untransduced Raji cells, Raji cells transduced with an empty vector (pSIP), and Raji cells overexpressing DR5.

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Expressing, Flow Cytometry, Labeling, Staining, Incubation, Fluorescence, Comparison, Control, Double Knockout, Transduction, Plasmid Preparation

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet:

Article Snippet: Mouse α‐human CD261 (DR4) Biotin (clone DJR1) , Miltenyi Biotec , Cat#130‐109‐084; RRID:AB_2656741.

Techniques: Generated, Recombinant, Control, Sequencing, Staining, Software, Selection, Enzyme-linked Immunosorbent Assay, Marker

Journal: Molecular Cell

Article Title: The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2

doi: 10.1016/j.molcel.2017.01.021

Figure Lengend Snippet:

Article Snippet: α-TRAIL-R1 , ProSci , Cat#PSC-1139-C100.

Techniques: Western Blot, Flow Cytometry, Recombinant, In Vivo, Red Blood Cell Lysis, Blocking Assay, Enzyme-linked Immunosorbent Assay, Proliferation Assay, cDNA Synthesis, Sequencing, Plasmid Preparation, Control, shRNA, Software

Journal: International journal of oncology

Article Title: A formulated red ginseng extract upregulates CHOP and increases TRAIL-mediated cytotoxicity in human hepatocellular carcinoma cells.

doi: 10.3892/ijo.2013.1964

Figure Lengend Snippet: Figure 4. The RGE sensitization to TRAIL-derived cell death occurs via death receptor 5 (DR5) upregulation in HCC cell lines. (A) DR5 protein levels were strongly dependent upon RGE concentration. (B) RGE‑induced upregulation of DR5, but not DR4, in HepG2 cells. (C) Upregulation of DR5 protein levels by RGE treatment in the different HCC cell lines.

Article Snippet: The following reagents were purchased and used according to the manufacturer's instructions: glutathione S-transferase (GST)-TRAIL and anti-DR5 antibodies were from Koma Biotechnologies (Seoul, Korea); anti-caspase 3, anti-PARP and anti-CHOP antibodies were from Cell Signaling Technology; anti-DR4 antibody was from Rockland; anti-tubulin antibody was from Abcam; anti-actin antibody, thapsigargin (Tg), necrostatin-1, NAC and BHA were from Sigma; and zVAD was from R&D Systems.

Techniques: Derivative Assay, Concentration Assay

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: CD13 inhibition enhances the effects of TRAIL in decreasing cell survival in tumor cells. (A) Expression levels of CD13, DR4, DR5, ERK1/2, and p-ERK1/2 in the indicated tumor cell lines, determined by Western blot analysis. (B) Analysis of the purification of TRAIL protein. M: Molecular weight protein markers (kDa); 1: TRAIL protein; 2: anti-TRAIL Western blot. (C) The tumor cell lines (A549, MCF-7, HT1080, and PANC-1) in 96-well plates were exposed to bestatin (0.01, 0.1, 0.5, 1, 1.25, 2.5, or 5 mg/mL) or WM15 (4, 12, or 20 μg/mL) for 48 h. (D) The indicated cell lines in 96-well plates were co-treated with TRAIL and bestatin for 48 h: A549, TRAIL 1.56–200 nmol/L, bestatin 0.01 or 0.1 mg/mL; MCF-7, TRAIL 1.56–200 nmol/L, bestatin 0.01 or 0.1 mg/mL; HT1080, TRAIL 0.039–5 μmol/L, bestatin 0.01 or 0.1 mg/mL; and PANC-1, TRAIL 0.039–5 μmol/L, bestatin 0.01 or 0.1 mg/mL. (E) The indicated cell lines were pretreated with WM15 (5 μg/mL) for 8 h and then co-treated with TRAIL (10 nmol/L) for an additional 48 h. The survival rates were normalized to the control group values (untreated cells). Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs . control, ## P < 0.01 vs . TRAIL.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Western Blot, Purification, Molecular Weight, Control

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: CD13 inhibition increases the expression of DR4 protein and cell surface DR4, and suppresses DR4 degradation. (A) The indicated cell lines were treated with bestatin (A549: 2 mg/mL, MCF-7: 1 mg/mL) for 4, 8, 12, or 24 h; (B) The indicated cell lines were treated with bestatin (A549: 1, 2, or 4 mg/mL, MCF-7: 0.5, 1, or 2 mg/mL) for 24 h. (C) The indicated cell lines were exposed to WM15 (5 μg/mL) for 24 h. (D) The indicated cell lines were exposed to CD13 siRNA (50 nmol/L) for 24 h. (E) The indicated cell lines were treated with bestatin (A549: 4 mg/mL, MCF-7: 2 mg/mL) for 24 h and then harvested for staining of DRs and subsequent flow cytometric analysis of cell surface DR4. The control cells were stained with a matched control FITC-conjugated IgG isotype antibody or FITC-conjugated anti-DR4 antibody, and the bestatin-treated cells were stained with FITC-conjugated anti-DR4 antibody. The MFI for each sample is indicated. (F) The indicated cell lines were exposed to bestatin (A549: 2 mg/mL, MCF-7: 1 mg/mL) for 24 h, and this was followed by the addition of 50 μg/mL CHX at different times. (G) Cells were pretreated with MG132 (10 μM) for 2 h and then received the indicated treatments for different times. Whole cell lysates were prepared from these cells and used to detect the levels of the indicated proteins with Western blot analysis. The results are plotted as the DR4 levels relative to those at time 0 of CHX treatment. The indicated proteins were quantified, and each was previously normalized to the level of GAPDH. Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs. control.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Staining, Control, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Loss of DR4 protects tumor cells against enhanced killing effects of TRAIL together with CD13 inhibition. (A) A549 and MCF-7 cells were exposed to 48 h pretreatment with 30 nmol/L DR4 siRNA, followed by 24 h treatment with TRAIL (50 nmol/L) and bestatin (0.5 mg/mL). (B) The expression of the indicated proteins was assessed by Western blot analysis after 36 h pretreatment with DR4 siRNA followed by 24 h treatment with TRAIL and bestatin at the indicated concentrations. Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs. TRAIL + bestatin + siCtrl.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Inhibition of ERK1/2 phosphorylation augments the cell death caused by TRAIL without up-regulating DR4. (A) The indicated cell lines in 96-well plates were pretreated with PD98059 (20 μmol/L) for 6 h followed by treatment with TPA (200 nmol/L) for 30 min and co-treatment with TRAIL (10 nmol/L) for an additional 48 h. The survival rates were normalized to the control group values (untreated cells). ** P < 0.01 vs. TRAIL + PD98059. (B) The indicated cell lines were treated with PD98059 (20 μmol/L) for 24 h, and then the whole cell lysates were prepared from the cells and used to detect the levels of phosphorylated and total ERK1/2 and DR4 by Western blot analysis. The indicated proteins were quantified, and each was previously normalized to the level of GAPDH. Data are means ± SD for 3 independent experiments. * P < 0.05, ** P < 0.01 vs. control.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Phospho-proteomics, Control, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Immunohistochemical staining to determine the expression of Ki67, DR4, and p-ERK1/2 in tumor tissues of nude mice treated with bestatin and/or TRAIL. (A) Cells with brownish yellow particles were considered Ki67 positive in HT1080 xenografts. The percentage of cells with positive staining for Ki67 expression was normalized to that in the control group. n = 6. ** P < 0.01 vs . control, ## P < 0.01 vs. TRAIL. The scale bar corresponds to 100 μm. The harvested tumors were lysed, and Western blot analysis was performed to detect the expression of DR4 (B), and phosphorylated and total ERK1/2 (C). The indicated proteins were quantified, and each was previously normalized to the level of tubulin. n = 3. * P < 0.05 vs. control. (D) Schematic presentation of the synergistic inhibitory mechanism.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Immunohistochemical staining, Staining, Expressing, Control, Western Blot

Journal: American Journal of Cancer Research

Article Title: PDE3A inhibitor anagrelide activates death signaling pathway genes and synergizes with cell death-inducing cytokines to selectively inhibit cancer cell growth

doi:

Figure Lengend Snippet: Effects of ANA on the expressions of cell cycle and cell death regulating genes. (A) Immunoblotting analyses of effects of ANA on cyclinD1 and P21 protein expression in the three types of cells. The cells were treated with ANA for the indicated time periods and the cell lysates were processed for immunoblotting with indicated antibodies. α-tubulin antibody was used as a control for equal protein loading. (B) Effects of ANA on the expressions of Bcl-XL and Bcl-2 proteins (left) and the effects of SLFN12 siRNA on the ANA-induced protein level changes of Bcl-XL and Bcl-2 (right). H4 cells were treated with or without ANA (100 nM), or with or without SLFN12 siRNA as indicate for 12 or 24 hours, and the cell lysates were processed for immunoblotting analyses using antibodies as indicated. (C and D) Effects of ANA on the expressions of TNF-α, DR4, and DR5 mRNAs and the effects of SLFN12 siRNA on the ANA-induced mRNA level changes of TNF-α, DR4, and DR5 in H4 cells. The H4 cells were treated with or without ANA (100nM), or with or without the SLFN12 siRNA as indicated for 18 h, and the cell lysates were processed and the mRNA levels of TNF-α (C), DR4, DR5 (D) were measured by quantitative RT-PCR analyses. (E) Effects of ANA on DR4 and DR5 protein expressions in the three types of cells. The cells were treated with ANA (100 nM) for indicated time periods and the cell lysates were processed for immunoblotting analyses using the indicated antibodies. Anti-tubulin antibody was used as a control for equal protein loading. (F) Effects of ANA or ANA plus SLFN12 siRNA on the activation of caspase 8 and caspase 9, protein expressions of DR4 and DR5, and cleavage of PARP. The H4 cells were treated with ANA, a combination of ANA and SLFN12 siRNA, for the indicated time periods, and the cells were processed for immunoblotting analyses using antibodies as indicated. Anti-tubulin antibody was used as a control for equal protein loading.

Article Snippet: Anti-PDE3A (#99236), anti-DR5 (#ab199357), anti-GAPDH (#8245) antibodies were purchased from Abcam; anti-Bcl-2 (#2876), anti-Bcl-XL (#2764), anti-Caspase-8 (#4790), anti-Caspase-9 (#9508), anti-Cleaved-PARP (#9541), anti-Cyclin D1 (#2978) antibodies were purchased from CST; anti-DR4 (#24063-1-AP) antibody was purchased from Proteintech; anti-α-tubulin (#5286) antibody was purchased from Santa Cruz; anti-PDE3B (#138892) antibody was purchased from Absin; anti-PARP (#556494) antibody was purchased from BD.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Activation Assay

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: TRAIL induces the assembly of apoptotic and non-apoptotic signaling complex(es). ( a ) Surface levels of Death Receptor 5 (anti-DR5-488), Death Receptor 4 (anti-DR4 PE), Decoy Receptor 1 (anti-DcR1 PE) and Decoy Receptor 2 (anti-DcR2-488) in BJELR cells. Surface levels of Death Receptor 4 in Hela cells and surface level of Decoy Receptor 1 in BJ cells were used as a positive control for DR4 and DcR1 labeling, respectively. Isotypic IgG1PE or Alexa 488 or IgG2B Alexa 488 labeling was used as a control for background fluorescence. ( b ) Western blots assessing the co-immunoprecipitation of indicated proteins (canonical DISC components) with Death Receptor 5 (immunoprecipitation target; DR5 IP), Death Receptor 4 (immunoprecipitation target; DR4 IP) or Decoy Receptor 2 (immunoprecipitation target; DcR2 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). NF, not detected. ( c ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with Death Receptor 5 (DR5 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with 1 μ g/ml TRAIL for 30 min (+). ( d ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with caspase-8 (immunoprecipitation target; C8 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). ( b – d ) Depicted images correspond to one representative experiment out of three independent biological replicates. Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Positive Control, Labeling, Control, Fluorescence, Western Blot, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors and TRAIL-receptor-binding proteins in the triggering of apoptosis. ( a ) Percentage of cells with high levels of cleaved PARP in populations transfected with pooled siRNAs targeting DR4 (DR4), DR5 (DR5), DcR2 (DcR2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged with TRAIL (6 h). ( b ) Percentage of cells with high levels of cleaved PARP in naïve (control) or TRAIL-challenged (6 h) DR5 knockout (DR5 KO) or DcR2 knockout (DcR2 KO) cells as compared with controls (ctrol). ( c ) Contour plot for cleaved PARP immunolabelling in DcR2 knockout (DcR2 KO) cells either unchallenged (control) or treated 6 h with TRAIL (TRAIL). Subpopulations displaying low (survivors, '1') and high (apoptotic, '2') levels of PARP cleavage in treated samples are depicted. Image shows one representative experiment out of three independent biological replicates. ( d ) Percentage of cells with high levels of cleaved PARP in control and DcR2 KO naïve cells (control) or control and DcR2 KO cells challenged with TRAIL for 16 h (TRAIL). ( e ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting FADD (FADD) , caspase-8 (casp-8), RIPK1 (RIPK1), TRAF2 (TRAF2) or cFlip (cFlip) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged for 6 h with TRAIL (TRAIL). ( f ) Percentage of cells with high levels of cleaved PARP in populations transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) or RIPK1 and TRAF2 (RIPK1+TRAF2) mRNAs or non-targeting scramble siRNAs at concentrations corresponding to single (30 nM; scr 30) or double (60 nM; scr 60) transfections, further left either untreated (control) or challenged during 6 h with TRAIL (TRAIL). Results obtained in conditions of pan-caspase inhibition (control+ zVAD and TRAIL+zVAD) as compared with basal conditions of cell response (control and TRAIL) are shown. ( g ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) , DR5 (DR5), RIPK1 and DR5 (RIPK1+DR5), TRAF2 and DR5 (TRAF2+DR5) mRNAs or non-targeting scramble siRNAs ('scr 30' and 'scr 60') further left either untreated (control) or challenged 6 h with TRAIL (TRAIL). ( a , b and d – g ) Mean±standard deviation (S.D.) from three independent biological replicates is shown. *** P- value <0.0005, ** P- value <0.005, * P- value <0.05. NS, not significant

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Binding Assay, Transfection, Control, Knock-Out, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors in the triggering of non-apoptotic and pro-survival pathways. ( a and b ) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38 and I κ B α in clonal populations of DcR2 knockout (DcR2 KO, a ), DR5 knockout (DR5 KO, b ) and control BJELR cells in basal conditions (0) or after treatment with TRAIL for the indicated time points. PARP cleavage is shown as a control for TRAIL treatment whereas DcR2 ( a ) or DR5 ( b ) is depicted as knockout controls. Images correspond to one representative experiment out of two independent biological replicates. ( c ) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38 and I κ B α in BJELR cells challenged with DR5-selective TRAIL-mimetic peptide M1D (10 μ M) as compared with controls (0). ( d ) Western blots displaying total protein and phosphorylation levels of Erk1/2, Akt, p38 and I κ B α in BJELR cells transfected either with siRNAs targeting DR4 and DcR2 (siDR4+siDcR2) mRNAs or non-targeting scramble siRNAs (scr) further left untreated (0) or challenged for 6 h with TRAIL. Uncropped image is shown as . Images correspond to one representative experiment out of at least two independent biological replicates. α -Tubulin, loading control

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Western Blot, Knock-Out, Control, Phospho-proteomics, Transfection

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Apoptotic and pro-survival TRAIL-induced signaling complex(es) are assembled at the plasma membrane. ( a ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 and TRAF2 with Death Receptor 5 (DR5 immunoprecipitation: DR5 IP). ( b and c ) Western blots assessing the co-immunoprecipitation of DR5, DR4, DcR2, FADD, cFlip and TRAF2 ( b ) as well as caspase-8 ( c ) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP). ( d ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DR5, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 with TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP). ( e ) Western blots assessing the co-immunoprecipitation of DR5, RIPK1 and TRAF2 with FADD (FADD immunoprecipitation: FADD IP). ( a–e ) Immunoprecipitations were performed from the purified plasma membrane (plasma membrane) obtained from either naïve cells (−) or cells treated with TRAIL (30 min '+' unless indicated otherwise in c ). Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control. Protein levels of indicated proteins at the plasma membrane are depicted as 'inputs'. Same blots are depicted as inputs for ( d ) and ( e ) as TRAF2 and FADD immunoprecipitation experiments were performed in parallel using the same protein input. Depicted images correspond to one representative experiment out of at least two independent biological replicates. N-Cadherin (N-Cdh) is shown as a plasma membrane marker. Controls for plasma membrane purification are depicted as . ND, not detected

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation, Purification, Control, Marker

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors in the formation of TRAIL-induced signaling platforms. ( a ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) with Death receptor 5 (DR5 immunoprecipitation: DR5 IP) in plasma membrane fractions obtained from DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( b ) Western blots assessing the co-immunoprecipitation of DR5 with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( c ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DcR2, FADD, caspase-8) with Death receptor 4 (DR4 immunoprecipitation: DR4 IP) in plasma membrane fractions obtained from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( d ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( e ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD, cFlip) with caspase-8 (caspase-8 immunoprecipitation: C8 IP) in whole-cell lysates from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( a – c and e ) Controls for plasma membrane purification are depicted as . ND, not detected; NA, not analyzed

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Knock-Out, Control, Purification

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

Article Title: MET inhibition downregulates DR4 expression in MET -amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

doi: 10.1016/j.neo.2021.06.006

Figure Lengend Snippet: Both pharmacological and genetic inhibition of MET decrease DR4 levels ( A-C ) including cell surface DR4 ( D and E ) in EGFR-TKI resistant HCC827 cells. A and B , The given cancer cell lines were treated with different concentrations of crizotinib for 8 h ( A ) or with 200 nM crizotinib for different times as indicated ( B ). C , Both HCC827/AR and HCC827/ER cells in 6-well plates were transfected with the indicated siRNAs for 48 h. After the aforementioned treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting analysis. D and E , The indicated cell lines were treated with 0.5 μM crizotinib for 12 h and then harvested for detection of cell surface DR4 with flow cytometry. The representative results are shown in D and average data (MFIs) from triplicate assays are presented in E as means ± SDs. The gray dot line open peaks in D represent DMSO-treated cells stained with a matched control PE-conjugated IgG isotype antibody. The black solid line open peaks show DMSO-treated cells stained with PE-conjugated anti-DR4 antibody. The filled peaks represent crizotinib (Criz)-treated cells stained with PE-conjugated anti-DR4 antibody.

Article Snippet: Alexa Fluor® 488-conjugated mouse anti-human DR4 (CD261) antibody (DR-4-02) was purchased from Bio-Rad Laboratories ( Hercules, CA ).

Techniques: Inhibition, Transfection, Western Blot, Flow Cytometry, Staining, Control

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

Article Title: MET inhibition downregulates DR4 expression in MET -amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

doi: 10.1016/j.neo.2021.06.006

Figure Lengend Snippet: Comparison of the effects of different MET inhibitors on modulating DR4 levels between MET -amplified EGFR-TKI resistant HCC827 cells and other NSCLC cell lines. The indicated cell lines were exposed to different concentrations of MET inhibitors as indicated ( A ) or treated with 1 μM of the given MET inhibitors ( B ) for 8 h. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of the indicated proteins.

Article Snippet: Alexa Fluor® 488-conjugated mouse anti-human DR4 (CD261) antibody (DR-4-02) was purchased from Bio-Rad Laboratories ( Hercules, CA ).

Techniques: Comparison, Amplification, Western Blot

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

Article Title: MET inhibition downregulates DR4 expression in MET -amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

doi: 10.1016/j.neo.2021.06.006

Figure Lengend Snippet: Crizotinib does not alter protein stability ( A ) and degradation ( B ) of DR4 in EGFR-TKI resistant HCC827 cells. A , The indicated cells were exposed to 0.5 μM crizotinib for 4 h followed by the addition of 10 μg/ml of CHX. At the indicated times post CHX, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH ImageJ software, normalized to actin and plotted as percentages of 0 time. The data are means +/- SEs of duplecated assays. B , The indicated cell lines were pre-treated with 10 μg/ml MG132 for 30 min and then co-treated with 0.5 μM MET inhibitors as indicated for an additional 5 h. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis.

Article Snippet: Alexa Fluor® 488-conjugated mouse anti-human DR4 (CD261) antibody (DR-4-02) was purchased from Bio-Rad Laboratories ( Hercules, CA ).

Techniques: Western Blot, Software

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

Article Title: MET inhibition downregulates DR4 expression in MET -amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

doi: 10.1016/j.neo.2021.06.006

Figure Lengend Snippet: Crizotinib decreases DR4 mRNA levels ( A ), suppresses AP-1-dependent DR4 promoter activity ( B ) and decreased the levels of c-Jun and p-c-Jun ( C ) in EGFR-TKI resistant HCC827 cells. A , The indicated cell lines were treated with 0.5 μM crizotinib for 8 h and then harvested for preparation of total cellular RNA and subsequent RT-PCR. B , HCC827/AR cells were transfected with the given DR4 reporter plasmids for 18 h and then treated with DMSO or 0.5 μM crizotinib for additional 10 h. The cells were then harvested for luciferase activity assay. The data are means ± SDs of triplicate determinations. C , The indicated cancer cell lines were treated with different concentrations of MET inhibitors as indicated for 8 h and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. LE, longer exposure. D , The pathways by which a MET inhibitor (METi) or its combination with an EGFR inhibitor (EGFRi) suppresses DR4 expression in EGFR-TKI resistant HCC827 cells were illustrated.

Article Snippet: Alexa Fluor® 488-conjugated mouse anti-human DR4 (CD261) antibody (DR-4-02) was purchased from Bio-Rad Laboratories ( Hercules, CA ).

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Western Blot, Expressing

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

Article Title: MET inhibition downregulates DR4 expression in MET -amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

doi: 10.1016/j.neo.2021.06.006

Figure Lengend Snippet: The combination of osimertinib and crizotinib augments induction of apoptosis ( A ) accompanied with enhanced suppression of ERK/c-Jun signaling and reduction of DR4 ( B ) in HCC827/AR cells and enhanced DR4 reduction in HCC827/AR xenografts ( C ). A and B , HCC827/AR cells were exposed to 100 nM osimertinib (Osim), different concentrations of crizotinib (Criz) as indicated and their respective combinations for 10 h ( B ) or 24 h ( A ) and then harvested for preparation of whole-cell protein lysates and subsequent Western blotting for detection of the indicated proteins. CF, cleaved from. C , The indicated proteins in different tissue lysates were detected with Western blotting. The tumor samples were from the experiment reported previously .

Article Snippet: Alexa Fluor® 488-conjugated mouse anti-human DR4 (CD261) antibody (DR-4-02) was purchased from Bio-Rad Laboratories ( Hercules, CA ).

Techniques: Western Blot

Journal: Oncogene

Article Title: Hypertonicity-enforced BCL-2 addiction unleashes the cytotoxic potential of death receptors

doi: 10.1038/s41388-018-0265-5

Figure Lengend Snippet: Changes in tonicity do not affect TRAIL-receptor expression and receptor-ligand interaction. a Cell surface expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was analyzed in HCT116 cells using flow cytometry. Data shown are representative of two experiments performed. b and c HCT116 cells were challenged with the indicated concentrations of b FLAG-TNC-TRAILmutR1 and c FLAG-TNC-TRAILmutR2 (both artificially cross-linked by adding anti-FLAG antibody) in the presence and absence of NaCl (75 mM). d HCT116 cells were treated with the indicated concentrations of the TRAIL-R2 targeting, agonistic antibody conatumumab (artificially cross-linked by adding protein G) in the presence and absence of NaCl (75 mM). For b – d , data points and mean ± SEM from three independent experiments are shown. e and f Equilibrium binding studies were performed by incubating SK-Mel-3 cells with the indicated concentrations of e GpL-FLAG-TNC-TRAIL and f GpL-FLAG-conatumumab as detailed in “Materials and methods.” Binding curves and R 2 values shown are representative of three experiments performed; K D values shown were calculated from three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, * p < 0.0001; n.s., not statistically significant; RLU, relative light units

Article Snippet: Antibodies used in the study were as follows: TRAIL-R1 (#854.853.020), TRAIL-R2 (#854.863.020) (Diaclone SAS, Besancon, France); cFLIP (#3210), caspase-8 (#9746), caspase-9 (#9502), cIAP1 (#7065), cIAP2 (#3130), XIAP (#2045), BID (#2002), BIM (#2933), BAX (#5023), BAK (#12105), PUMA (#12450), BCL-xL (#2764), MCL-1 (#5453), and BCL-W (#2724) (Cell Signaling, Beverly, MA, USA); tubulin (#MS-581) (Dunnlab, Asbach, Germany); conformation-specific [ ] BAX (6A7, #sc-23959), NOXA (114C307, #sc-56169) (Santa Cruz, Santa Cruz, CA, USA); FLAG (M2, #F3165) (Sigma); NFAT5 (#PA1–023) (Thermo Fisher).

Techniques: Expressing, Flow Cytometry, Binding Assay

Journal: Oncogene

Article Title: Hypertonicity-enforced BCL-2 addiction unleashes the cytotoxic potential of death receptors

doi: 10.1038/s41388-018-0265-5

Figure Lengend Snippet: TRAIL-induced receptor signaling complex formation and caspase-8 activation is equally effective under iso- and hypertonic conditions. a TRAIL-R1/-R2 signaling complexes were induced in HCT116 cells by stimulation with Fc-FLAG-scTRAIL (1 μg/ml) for the indicated periods of time in the presence and absence of NaCl (75 mM). Proteins associated with Fc-FLAG-scTRAIL were immunoprecipitated using protein G agarose and were analyzed together with the corresponding lysates by western blotting for the presence of the indicated proteins. Data shown are representative of two experiments performed. b and c HCT116 cells were treated with KillerTRAIL (32 ng/ml) in the presence and absence of NaCl (75 mM) for the indicated periods of time. Caspase-8 activity was assessed using the fluorogenic substrate (IETD) 2 -R110. Please note that different concentrations of recombinant TRAIL required for caspase activity assays and co-immunoprecipitation experiments could affect the kinetics of caspase-8 activation. Shown are data points and mean ± SEM from three independent experiments. d HCT116 and SK-Mel-3 cells were challenged with KillerTRAIL (32 ng/ml) for 9 h in the presence and absence of NaCl (75 mM). After washing and lysis, western blot analyses were performed with antibodies specific for the indicated proteins. Detection of tubulin served as a loading control. RFU, relative fluorescence units

Article Snippet: Antibodies used in the study were as follows: TRAIL-R1 (#854.853.020), TRAIL-R2 (#854.863.020) (Diaclone SAS, Besancon, France); cFLIP (#3210), caspase-8 (#9746), caspase-9 (#9502), cIAP1 (#7065), cIAP2 (#3130), XIAP (#2045), BID (#2002), BIM (#2933), BAX (#5023), BAK (#12105), PUMA (#12450), BCL-xL (#2764), MCL-1 (#5453), and BCL-W (#2724) (Cell Signaling, Beverly, MA, USA); tubulin (#MS-581) (Dunnlab, Asbach, Germany); conformation-specific [ ] BAX (6A7, #sc-23959), NOXA (114C307, #sc-56169) (Santa Cruz, Santa Cruz, CA, USA); FLAG (M2, #F3165) (Sigma); NFAT5 (#PA1–023) (Thermo Fisher).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Activity Assay, Recombinant, Lysis, Control, Fluorescence