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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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 CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

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

Journal: The American Journal of Pathology

Article Title: Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells

doi: 10.1016/s0002-9440(10)64220-4

Figure Lengend Snippet: Figure 7. A: Flow cytometric analysis for TRAIL receptors (DR4, DR5, DcR1, and DcR2, shaded peaks) in SW579 cells treated with or without IGF-1 (100 ng/ml) for 8 hours revealed no changes. Control antibody staining appears as unshaded peaks. B: Treatment of SW579 cells with IGF-1 (100 or 300 ng/ml) for 8 hours up-regulated the apoptosis inhibitors FLIP, cIAP2, XIAP, and survivin, but not cIAP1 or the anti-apoptotic members of the Bcl-2 family Bcl-2, Bcl-xL, A1, and Mcl-1. IGF-1 also down-regulated the proapoptotic Bax.

Article Snippet: Recombinant human TRAIL was obtained from Immunex Corporation (Seattle, WA), in a leucine zipper (LZ) form that promotes and stabilizes the formation of trimers, as previously reported;13 for comparison, several experiments were repeated using the recombinant Apo2L form from Genentech Inc. (South San Francisco, CA).20 The goat polyclonal antibodies for DR4, DR5, DcR1, and mouse monoclonal antibody for tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA); the anti-DcR2 rabbit polyclonal antibody was from Imgenex (San Diego, CA); cycloheximide, geldanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St Louis, MO); IGF-1, bFGF, EGF, IFN- , and TNF- were from R&D Systems (Minneapolis, MN; bisindolylmaleimide (BIM) III and wortmannin were from Calbiochem (La Jolla, CA); rabbit polyclonal antibody for caspase-10 was from Research Diagnostics Inc. (Flanders, NJ); the IGF-1 receptor neutralizing antibody aIR3 was from Oncogene Research (Cambridge, MA); and the enhanced chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham (Arlington Heights, IL).

Techniques: Control, Staining

Journal: The American Journal of Pathology

Article Title: Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells

doi: 10.1016/s0002-9440(10)64220-4

Figure Lengend Snippet: Figure 11. A and B: Flow cytometric analysis of TRAIL receptors DR4 (A) and DR5 (B) after a 48-hour treatment with or without IFN- (500 IU/ml) or TNF- (50 ng/ml) in SW579 cells. Control antibody staining is also shown. C: Evaluation of the protein levels of caspase-8, caspase-10, caspase-3, FLIP, and TRAIL in SW579 cells after a 48-hour treatment with or without IFN- (500 IU/ml) or TNF- (50 ng/ml). IFN- (500 IU/ml) up-regulated caspase-8 and TNF- (50 ng/ml) and up-regulated caspases-10 and -3. Additionally, TNF- induced the expression of TRAIL itself.

Article Snippet: Recombinant human TRAIL was obtained from Immunex Corporation (Seattle, WA), in a leucine zipper (LZ) form that promotes and stabilizes the formation of trimers, as previously reported;13 for comparison, several experiments were repeated using the recombinant Apo2L form from Genentech Inc. (South San Francisco, CA).20 The goat polyclonal antibodies for DR4, DR5, DcR1, and mouse monoclonal antibody for tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA); the anti-DcR2 rabbit polyclonal antibody was from Imgenex (San Diego, CA); cycloheximide, geldanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St Louis, MO); IGF-1, bFGF, EGF, IFN- , and TNF- were from R&D Systems (Minneapolis, MN; bisindolylmaleimide (BIM) III and wortmannin were from Calbiochem (La Jolla, CA); rabbit polyclonal antibody for caspase-10 was from Research Diagnostics Inc. (Flanders, NJ); the IGF-1 receptor neutralizing antibody aIR3 was from Oncogene Research (Cambridge, MA); and the enhanced chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham (Arlington Heights, IL).

Techniques: Control, Staining, Expressing

Journal: International journal of oncology

Article Title: Reversine induces cell cycle arrest and apoptosis via upregulation of the Fas and DR5 signaling pathways in human colorectal cancer cells.

doi: 10.3892/ijo.2019.4746

Figure Lengend Snippet: Figure 2. Reversine induces apoptosis in human colorectal cancer cells. SW480 and HCT116 cells were exposed to different concentrations of reversine for 24 h. (A) Apoptotic DNA fragmentation was analyzed by agarose gel electrophoresis. (B) Treated cells were stained with Annexin V and the number of apop totic cells was evaluated. (C) Protein levels were detected by western blotting. GAPDH was used as a loading control. Cas, caspase; PARP, poly(ADP‑ribose) polymerase; SMAC/DIABLO, second mitochondria‑derived activator of caspase/direct inhibitor of apoptosis‑binding protein with low pI; DR5, death receptor 5; 7‑AAD, 7‑amino‑actinomycin D.

Article Snippet: Fas and DR5 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Agarose Gel Electrophoresis, Staining, Western Blot, Control

Journal: International journal of oncology

Article Title: Reversine induces cell cycle arrest and apoptosis via upregulation of the Fas and DR5 signaling pathways in human colorectal cancer cells.

doi: 10.3892/ijo.2019.4746

Figure Lengend Snippet: Figure 4. Pan‑caspase inhibitor attenuates reversine‑induced inhibition of cell growth and induction of apoptosis in human colorectal cancer cells. SW480 and HCT116 cells were pretreated with 20 µM of the pan‑caspase inhibitor Z‑VAD‑FMK for 2 h and then exposed to the indicated concentrations of reversine for 24 h. (A) Cell morphology was observed under a fluorescence microscope following Calcein‑acetomethoxy solution staining. (B) Treated cells were stained with Annexin V and the number of apoptotic cells was counted. (C) Protein levels were detected by western blotting. GAPDH was used as a loading control. Cas, caspase; PARP, poly(ADP‑ribose) polymerase; DR5, death receptor 5; 7‑AAD, 7‑amino‑actinomycin D.

Article Snippet: Fas and DR5 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Inhibition, Fluorescence, Microscopy, Staining, Western Blot, Control

Journal: International journal of oncology

Article Title: Reversine induces cell cycle arrest and apoptosis via upregulation of the Fas and DR5 signaling pathways in human colorectal cancer cells.

doi: 10.3892/ijo.2019.4746

Figure Lengend Snippet: Figure 5. Impact of reversine on Fas and DR5 signaling pathways in human colorectal cancer cells. SW480 and HCT116 cells were transfected with Fas and DR5 siRNA for 24 h and then exposed to the indicated concentrations of reversine for 24 h. (A) Treated cells were stained with Annexin V and the number of apoptotic cells was counted. (B) Treated cells were stained with PI and subjected to flow cytometric analysis to determine the cell distribution at each phase of cell cycle. (C) Protein levels were detected by western blotting. GAPDH was used as a loading control. siRNA, small interfering RNA; SS, scrambled siRNA, DS, death receptor 5 siRNA; FS, Fas siRNA; Cas, caspase; PARP, poly(ADP‑ribose) polymerase; DR5, death receptor 5; PI, propidium iodide; 7‑AAD, 7‑amino‑actinomycin D.

Article Snippet: Fas and DR5 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Protein-Protein interactions, Transfection, Staining, Western Blot, Control, Small Interfering RNA

Journal: Cancer cell

Article Title: A Single Agent Dual Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer

doi: 10.1016/j.ccell.2018.07.005

Figure Lengend Snippet: (A) Healthy tissues are generally non-responsive to agonist DR5 therapy because they express no or very low level of FOLR1, thus DR5 oligomerization and activation is very minimal.

Article Snippet: TRAIL-R2 receptor in oligomerization was determined using immunoblotting assays (cell signaling Rabbit mAb, 8074).

Techniques: Activation Assay

Journal: Molecular Cancer Therapeutics

Article Title: PKCδ Regulates Death Receptor 5 Expression Induced by PS-341 through ATF4–ATF3/CHOP Axis in Human Lung Cancer Cells

doi: 10.1158/1535-7163.mct-12-0602

Figure Lengend Snippet: Figure 1. PS-341 (A) enhances CHOP expression in a dose-dependent and a time-dependent manner (B), and inhibiting CHOP expression by siRNA decreases PS-341–induced DR5 upregulation and apoptosis (C and D). A, the chemical structure of PS-341. B, the indicated lung cancer cell lines were treated with 0, 100, 200 nmol/L PS-341 for 24 hours or treated with 50 nmol/L PS-341 for the indicated time. H460, A549 (C), and Calu-1 (C and D) cells were seeded in 6-well plates and on the second day transfected with control (Ctrl) or CHOP siRNA. Forty-eight hours after the transfection, cells were treated with the indicated concentration of PS-341 for 16 hours (C) or 24 hours (D). Then the cells were harvested and prepared for Western blot analysis (C) or for detection of apoptotic cells using Annexin V/7-AAD staining (D). CF, cleaved form.

Article Snippet: DR5 antibody was purchased from ProSci.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Staining

Journal: Molecular Cancer Therapeutics

Article Title: PKCδ Regulates Death Receptor 5 Expression Induced by PS-341 through ATF4–ATF3/CHOP Axis in Human Lung Cancer Cells

doi: 10.1158/1535-7163.mct-12-0602

Figure Lengend Snippet: Figure 6. PS-341 stimulates the cleavage of PKCd in a dose-dependent and time-dependent manner (A), and silencing of PKCd expression by siRNA attenuates DR5 induction (B) and apoptosis induced by PS-341 (C and D). The indicated lung cancer cell lines were treated with 0, 100, 200 nmol/L PS-341 for 24 hours or treated with 50 nmol/L PS-341 for the indicated time (A). H157 (B and C) and Calu-1 (B-D) cells were seeded in 6-well plates and on the second day transfected with control (Ctrl) or PKCd siRNA. Forty-eight hours after the transfection, cells were treated with 100 nmol/L PS-341 for 16 hours (B and C) or 24 hours (D). Then the cells were harvested and prepared for Western blot analysis (B and C) or for detection of apoptotic cells using Annexin V/7-AAD staining (D). CF, cleaved form.

Article Snippet: DR5 antibody was purchased from ProSci.

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