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: Nature cell biology

Article Title: Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay

doi: 10.1038/s41556-020-00618-1

Figure Lengend Snippet: a, As in Fig. 4a, dot plots of RT-qPCR quantitations of the specified mRNA, normalized to the level of its pre-mRNA, using lysates from healthy (i.e. normal) or FXS lymphoblasts. Red bars represent means with S.D, where n = 6 independent biological cell lines. P values were calculated in comparisons of normal vs. FXS samples (two-sided Wilcoxon rank-sum test). b, as in a, but normalized to the level of ß-actin mRNA. Red bars, means with S.D., n = 6 independent biological cell lines. P values were calculated in comparisons of normal vs. FXS samples (two-sided Wilcoxon rank-sum test). c, Bright-field and IF images of iPSCs from a representative normal or a representative FXS patient-derived cell line for validation purposes. For IF, FXS iPSCs were stained for TRA-1-60 (green) or OCT4 (red), each of which is a pluripotent marker. Scale bar, 200 μm. Results are representative of 3 independent biological replicates. d, as in Fig. 4b, but normalized to the level of ß-actin mRNA. Means with S.D., where n = 3 biologically independent replicates. (***) P < 0.001 are relative to Normal #1 samples (one-way ANOVA and Dunnett’s multiple comparison test). e, As in Fig. 4b but for isogenic iPSCs + or − CGG repeat expansion. Means with S.D., where n = 3 independent biological replicates. P values were calculated in comparisons of + vs. − CGG repeat expansion (two-sided t-test). f, Western blots of lysates of normal and FXS iPSCs from Fig. 4b. Results are representative of 3 independent biological replicates. g, Protocol of neural differentiation − or + an NMD inhibitor. h, Quantitations of western blots shown in Fig. 4c. i, FXS iPSC-derived neurons exhibit deficient neurite formation on day 7 of differentiation. βIII-tubulin staining of normal and FXS neurons derived from iPSCs was used to assess neurite formation, average neurite length, neurite ramification, and neuronal differentiation efficacy. Means with S.D., where n = 8 independent biological replicates. P values were calculated in comparisons of normal vs. FXS samples (two-sided t-test). j, Quantitations of IF shown in Fig. 4d. Results are means with S.D., where n = 5 independent biological replicates. P values were calculated in comparisons of normal vs. FXS samples (two-sided t-test). Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 7.

Article Snippet: Coverslips were blocked with 3% bovine serum albumin (Rockland Immunochemicals) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 30 min at room temperature, washed once with TBS-T and incubated overnight at 4 °C in primary antibody that had been diluted in TBS-T using the following antibodies (see Supplementary Table 6 ): anti-p-UPF1 S1116 (1:250), anti-p-UPF1 S1089 (1:250), anti-UPF1 (1:500), anti-FMRP (1:250), anti-TRA-1–60 (1:500), anti-OCT4 (1:500), anti-MAP2 (1/250), anti-β3-Tubulin/TUJ1 (1:500) and anti-BRN2/POU3F2 (1:250).

Techniques: Quantitative RT-PCR, Derivative Assay, Staining, Marker, Western Blot

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

Journal: Oncology letters

Article Title: YM155 sensitizes HeLa cells to TRAIL-mediated apoptosis via cFLIP and survivin downregulation.

doi: 10.3892/ol.2020.11933

Figure Lengend Snippet: Figure 3. Combination treatment downregulates mRNA and protein levels of cFLIP and survivin. Reverse transcription‑quantitative PCR was used to assess the mRNA expression levels of (A) cFLIP, (B) DR5 and (C) survivin following treatment with 25 ng/ml TRAIL and 25 nM YM155 for 24 h. Data are presented as the mean and standard deviation of three independent experiments. One‑way ANOVA followed by Tukey's post hoc test was used, and P‑values are indi cated. (D) Western blotting was performed to assess protein expression levels of cFLIP, DR5 and survivin after treatment with 25 ng/ml TRAIL and 25 nM YM155 for 24 h. GAPDH and β‑tubulin were used as the loading controls. cFLIP, CASP8 and FADD like apoptosis regulator; DR5, death receptor 5; TRAIL, tumor necrosis factor‑related apoptosis inducing ligand.

Article Snippet: Western blot and immunofluorescence analyses were performed using antibodies against cleaved‐poly (ADP‐ribose) polymerase (PARP; dilution, 1:1,000; cat. no. 5625S) from Cell Signaling Technology, Inc., cFLIP (dilution, 1:1,000; cat. no. 10394‐1‐AP) and DR5 (dilution, 1:1,000; cat. no. 15497‐1‐AP) from ProteinTech Group, Inc., γH2A histone family member X (γH2AX; dilution, 1:1,000 for western blot and 1:100 for immunofluorescence; cat. no. 05‐636) from EMD Millipore, cleaved caspase‐3 (dilution, 1:100 for immunofluorescence; cat. no. 9661S) from Cell Signaling Technology, Inc., GAPDH (dilution, 1:2,000; cat. no. sc‐32233) and β‐tubulin (1:1,000; cat. no. sc‐5274) from Santa Cruz Biotechnology, Inc., and HRP‐conjugated secondary anti‐mouse (1:10,000; cat. no. 31430) and anti‐rabbit (1:10,000; cat. no. 31460) antibodies from Thermo Fisher Scientific, Inc., and fluorescence‐conjugated secondary anti‐ bodies goat anti‐mouse IgG (H+L) Alexa Fluor 488 (1:250; cat. no. A32723) and goat anti‐rabbit IgG (H+L) Alexa Fluor 488 (1:250; cat. no. A27034) from Invitrogen, Thermo Fisher Scientific, Inc. Western blotting.

Techniques: Expressing, Standard Deviation, Western Blot