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: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Cell viability assay in prostate cancer cell lines after docetaxel and TRAIL treatment. Cells are exposed for 24 hours, and cell viability is determined using the MTT assay (A, B). Flow cytometry analysis of DU 145 shows that a combination of docetaxel 2 nM and TRAIL 100 ng/mL increases the proportion of Annexin V(+)/PI(+) (C). The expressions of EZH2 and DR4 in prostate cancer cell lines are shown. A WB assay is performed with specific antibodies against EZH2, DR4, DR5, and β-actin (D). After docetaxel treatment for 24 hours, the EZH2 level is decreased and the DR4 level is increased in DU 145 cells (E). The cell viability test is performed with a combination of docetaxel and TRAIL with or without the DR4 antagonist antibody (F). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, ns: not significant, PI: propidium iodide, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot. *** p<0.001.

Article Snippet: TRAIL receptor inhibitors (neutralizing antibodies) and anti-human DR4 (ALX-804-297A; Enzo Life Sciences, Farmingdale, NY, USA) were always added 1 hour before the addition of TRAIL.

Techniques: Viability Assay, MTT Assay, Flow Cytometry, Western Blot

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Cell viability assay in prostate cancer cell lines after EZH2 inhibitors and TRAIL treatment. Cells are exposed for 48 hours. Cell viability is determined using the MTT assay (A, B). A WB assay is performed with specific antibodies against EZH2, DR4, DR5, and β-actin in prostate cancer cell lines (C-E). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: TRAIL receptor inhibitors (neutralizing antibodies) and anti-human DR4 (ALX-804-297A; Enzo Life Sciences, Farmingdale, NY, USA) were always added 1 hour before the addition of TRAIL.

Techniques: Viability Assay, MTT Assay, Western Blot

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Expressions of EZH2 and E2F1 in DU 145 cells. WB assay shows decreases of EZH2 and E2F1 expressions after docetaxel treatment (A). Using knockdown of E2F1, the expressions of E2F1 and EZH2 are decreased (B), and TRAIL (100 ng/mL) treatment causes significant cell death in siE2F-1 DU 145 cells ( *** p<0.001) (C). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, ns: not significant, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot.

Article Snippet: TRAIL receptor inhibitors (neutralizing antibodies) and anti-human DR4 (ALX-804-297A; Enzo Life Sciences, Farmingdale, NY, USA) were always added 1 hour before the addition of TRAIL.

Techniques: Western Blot

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: MSP analysis is performed to evaluate the methylation status (A, B). ChIP assay is used to assess the relationship of EZH2 and the DR4 promoter in LNCap-LN3 and DU 145 cells (C). After docetaxel or DZNep treatment, the relationship of EZH2 and the promoter lesion of DR4 or DR5 is evaluated (D). ChIP: chromatin immunoprecipitation, DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, IgG: immunoglobulin G, MSP: methylation-specific polymerase chain reaction, ns: not significant, PCR: polymerase chain reaction. * p<0.05, ** p<0.01.

Article Snippet: TRAIL receptor inhibitors (neutralizing antibodies) and anti-human DR4 (ALX-804-297A; Enzo Life Sciences, Farmingdale, NY, USA) were always added 1 hour before the addition of TRAIL.

Techniques: Methylation, Chromatin Immunoprecipitation, Polymerase Chain Reaction

Journal: Biochemistry and Biophysics Reports

Article Title: Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

doi: 10.1016/j.bbrep.2021.100951

Figure Lengend Snippet: Association between C2GnT expression and NK cell toxicity via the TRAIL-DR pathways. (A) Viability of control, C2GnT KO Jar and BeWo cells using MTS assay. Data were obtained from three individual experiments and each condition was assessed by triplicates. Mean ± SD of relative cell counts are shown. (B) Cell viability of control, C2GnT KO Jar and BeWo cells treated with endo-β-galactosidase with 1 μg/mL TRAIL for 24 h. Data were obtained from three individual experiments and each condition was assessed by triplicates. Mean ± SD of cytotoxicity rates are shown. n.s., not significant. (C) Representative western blot demonstrating MUC1 and DR4 protein expression in control, C2GnT KO Jar and BeWo cells. LEL immunoprecipitates were subjected to western blotting with anti-MUC1.

Article Snippet: Western blotting was performed as previously described [ ] using the anti-C2GnT1 (Wako), anti-MICA/B (Biolegend), anti-death receptor 4 (DR4) (Funakoshi, Tokyo, Japan), and anti-mucin1 (MUC1) (Abcam, Cambridge, UK) antibodies.

Techniques: Expressing, Control, MTS Assay, Western Blot

Journal: International Journal of Oncology

Article Title: Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

doi: 10.3892/ijo.2013.1976

Figure Lengend Snippet: Activation of TRAIL-death receptors DR4 and DR5 by Asp. SW480 and SW620 cells were treated with Asp (80 μ g/ml) for 24 and 48 h. The expression of DR4 and DR5 receptors were analyzed by flow cytometry after staining with respective FITC-conjugated antibodies (details in Materials and methods). In the upper panel representative FACS histograms are shown, a shift to the right of fluorescence intensity (green line) corresponds to an increase of DR4 or DR5 receptor expression at the cell surface. In the lower panel data are presented as cytometer histogram plots representing the percentage of cells expressing DR4 or DR5 receptors. Data are the mean value ± SE of three separate experiments. For each cell line and time period: Asp vs. controls, * P<0.05.

Article Snippet: Cells were treated with Asp (80 μ g/ml) and harvested by trypsinization at 24 and 48 h. Cell pellets were washed with PBS and incubated with FITC-conjugated mouse anti-human TRAIL receptors DR4 and DR5 (Alexis Biochemicals), or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences) for 30 min at 4°C in the dark.

Techniques: Activation Assay, Expressing, Flow Cytometry, Staining, Fluorescence, Cytometry

Journal: International Journal of Oncology

Article Title: Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

doi: 10.3892/ijo.2013.1976

Figure Lengend Snippet: Involvement of TRAIL DR4/DR5 receptors in Asp-triggered cell death. SW480 and SW620 cells were treated with Asp (80 μ g/ml) for 24 and 48 h. (A) Relative expression of DR4 and DR5 mRNA expression levels in SW480 and SW620 cells. Total RNA was isolated from controls and Asp-treated cells and qRT-PCR was performed in triplicate as detailed in Materials and methods. The mRNA level from control cells was used as an external reference and the level of β-actin mRNA was used as an internal reference to normalize the data. The mRNA fold-changes (mRNA relative expression) were expressed relative to the corresponding mRNA mean value found in control cells. Data are presented as the mean ± SE. Asp vs. controls * P<0.01. (B) SW480 and SW620 cells were treated with Asp (80 μ g/ml) ± DR4/Fc and DR5/Fc chimeric proteins (@DR4/DR5, 100 ng/ml) exhibiting a dominant-negative effect by blocking the endogenous receptors for 24 and 48 h. SW480 and SW620 cells were then harvested and stained with propidium iodide for the measurement of hypodiploid bodies and analyzed by flow cytometry as detailed in Materials and methods. Data are the mean value ± SE of at the least three separate experiments. For 24 or 48 h, columns not sharing the same superscript letter differ significantly, P<0.05.

Article Snippet: Cells were treated with Asp (80 μ g/ml) and harvested by trypsinization at 24 and 48 h. Cell pellets were washed with PBS and incubated with FITC-conjugated mouse anti-human TRAIL receptors DR4 and DR5 (Alexis Biochemicals), or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences) for 30 min at 4°C in the dark.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Dominant Negative Mutation, Blocking Assay, Staining, Flow Cytometry

Journal: International Journal of Oncology

Article Title: Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

doi: 10.3892/ijo.2013.1976

Figure Lengend Snippet: Asp activates apoptotic cell death in the colonic mucosa of AOM-injected rats. (A) Changes in the mRNA expression levels of various cell death biomarkers in the colonic mucosa of AOM-injected rats (n=8) and Asp treated AOM-injected rats (n=8). Real-time PCR was performed in triplicate wells. For further details see legend of . (B) Western blot analysis of the pro-apoptotic DR5 receptor and of TRAIL protein expression in the mucosal samples of AOM-injected control rats (AOM) and Asp-treated AOM-injected rats, β-actin was used as an internal control. (C) Caspase-3 activity in the colonic mucosal samples of AOM-injected control rats and Asp treated AOM-injected rats. For details see Materials and methods. Data are presented as the mean ± SE, Asp-treatment vs. control, * P<0.01.

Article Snippet: Cells were treated with Asp (80 μ g/ml) and harvested by trypsinization at 24 and 48 h. Cell pellets were washed with PBS and incubated with FITC-conjugated mouse anti-human TRAIL receptors DR4 and DR5 (Alexis Biochemicals), or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences) for 30 min at 4°C in the dark.

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay