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: 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: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT activates TRAIL-death receptor signaling in VHL -deficient ccRCC cells (A) Volcano plot of SGI1027-induced and -repressed genes in RCC10 cells ( n = 2 biological replicates). FC, fold change. (B) Biocarta pathway enrichment analysis of SGI1027-induced genes in RCC10 cells. (C) RT-qPCR analysis of TNFSF10 , TNFRSF10A , TNFRSF10B , and TNFRSF10D mRNA levels in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (D and E) Immunoblot analysis of TRAIL, DR4, DR5, DcR2, pro-caspase-10, and cleaved caspase-10 (C-caspase-10) proteins in isogenic RCC10 cells treated with vehicle, SGI1027 (D and E, n = 2 biological replicates), MS1129 (E, n = 2 biological replicates), or decitabine (E, n = 2 biological replicates) for 2 or 7 days. (F) Global m5C levels in RCC10 cells treated with vehicle or SGI1027 for 2 days by ELISA assay ( n = 3 biological replicates). (G–J) MeDIP-qPCR assay in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (K–M) DNMT1, DNMT3A, and DNMT3B ChIP-qPCR assay in RCC10 cells ( n = 3 biological replicates). (N) Scheme of dDNMT-activated apoptotic pathway. Data represent mean ± SEM. p value was determined by bioinformatics with edgeR (A) or gene set enrichment analysis (B), unpaired 2-tailed Student’s t test (C and F), two-way ANOVA with Tukey’s test (G–I), and one-way ANOVA with Dunnett’s test (K–M). See also and .

Article Snippet: The following antibodies were used: anti-HIF-1α antibody (A300-286A, Bethyl Laboratories), anti-HIF-2α antibody (home-made), anti-DNMT1 antibody (24206-1-AP, Proteintech), anti-DNMT3A antibody (20954-1-AP, Proteintech), anti-DNMT3B antibody (26971-1-AP, Proteintech), anti-DNMT3L antibody (sc-393603, Santa Cruz), anti-cleaved-caspase-3 antibody (9661S, Cell Signaling Technology), anti-cleaved-caspase-7 antibody (8438S, Cell Signaling Technology), anti-PARP1 antibody (9542S, Cell Signaling Technology), anti-caspase-10 antibody (14311-1-AP, Proteintech), anti-caspase-8 antibody (13423-1-AP, Proteintech), anti-TRAIL antibody (27064-1-AP, Proteintech), anti-DR4 antibody (24063-1-AP, Proteintech), anti-DR5 antibody (69400S, Cell Signaling Technology), anti-DcR2 antibody (16781-1-AP, Proteintech), anti-β-actin (81115-1-RR, Proteintech), and anti-α-tubulin (66031-1-Ig, Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Methylated DNA Immunoprecipitation, ChIP-qPCR

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT specifically kills patient-derived VHL -deficient ccRCC in mice (A) Tumor growth curves of VHL -deficient UTSW-PDX206, UTSW-PDX258, UTSW-PDX490, and UTSW-PDX26 in mice treated with vehicle (Veh) or SGI1027 for 10 days. (B) Tumor growth curves of VHL -WT UTSW-PDX416 and UTSW-PDX143 in mice treated with vehicle or SGI1027 for 10 days. (C) Kaplan-Meier survival curve of UTSW-PDX490-bearing mice ( n = 10 biological replicates). (D and E) Global m5C levels in UTSW-PDX206 (D) or UTSW-PDX258 (E) tumors harvested from mice after treatments by ELISA assay ( n = 5 biological replicates). (F) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, procaspase-10, C-caspase-3, and C-caspase-7 proteins in UTSW-PDX258 tumors harvested from mice after treatments ( n = 5 biological replicates). (G) Representative C-caspase-3 IHC in UTSW-PDX258 tumors. Scale bar, 100 μm. (H) Quantification of C-caspase-3-positive cells in (G) ( n = 5 biological replicates). (I) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, C-caspase-3, and VHL proteins in UTSW-PDX416 tumors harvested from mice after treatments ( n = 5 biological replicates). (J) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, and procaspase-10 proteins in UTSW-PDX206, UTSW-PDX258, and UTSW-PDX26 tumors ( n = 4–5 biological replicates). Data represent mean ± SEM. p value was determined by two-way ANOVA with Tukey’s test (A), log rank test (C), and unpaired 2-tailed Student’s t test (D, E, and H). See also and ; .

Article Snippet: The following antibodies were used: anti-HIF-1α antibody (A300-286A, Bethyl Laboratories), anti-HIF-2α antibody (home-made), anti-DNMT1 antibody (24206-1-AP, Proteintech), anti-DNMT3A antibody (20954-1-AP, Proteintech), anti-DNMT3B antibody (26971-1-AP, Proteintech), anti-DNMT3L antibody (sc-393603, Santa Cruz), anti-cleaved-caspase-3 antibody (9661S, Cell Signaling Technology), anti-cleaved-caspase-7 antibody (8438S, Cell Signaling Technology), anti-PARP1 antibody (9542S, Cell Signaling Technology), anti-caspase-10 antibody (14311-1-AP, Proteintech), anti-caspase-8 antibody (13423-1-AP, Proteintech), anti-TRAIL antibody (27064-1-AP, Proteintech), anti-DR4 antibody (24063-1-AP, Proteintech), anti-DR5 antibody (69400S, Cell Signaling Technology), anti-DcR2 antibody (16781-1-AP, Proteintech), anti-β-actin (81115-1-RR, Proteintech), and anti-α-tubulin (66031-1-Ig, Proteintech).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot