Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: A variety of ER stressors alter DR5 disulfide bonding. A, upper panel. ERp44-deficient HepG2 cells into which vector, wild type or catalytically null ERp44 were reintroduced were treated as indicated for 24 h and analyzed by non-reducing immunoblot. A, lower panel. Expanded region of the DR5 immunoblot showing altered DR5 disulfide bonding in the Thapsigargin/PERKi combination treatment. B. Non-reducing immunoblot of MDA-MB-468 cells treated as indicated for 24 h. C. MDA-MB-468 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. D. Protein synthesis assays of cells pre-treated for 24 h as indicated before protein synthesis was measured by 3 H-Leucine incorporation over a 2 h pulse. Data are plotted as the average (N = 6), with error bars representing standard deviation. E. HepG2 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. F. Control and PERK knockout HepG2 cells were treated for 24 h as indicated and subjected to non-reducing immunoblot analysis. G. Non-reducing immunoblot analysis of the indicated cell lines treated as specified for 24 h. Note the DR5 oligomerization in A431 cells when Thapsigargin, Tunicamycin, or dFtcyDTDO are combined with PERK inhibition. H. Non-reducing immunoblot analysis of WM793 cells treated as specified for 24 h. Unless otherwise specified, the following concentrations of compounds were used in A-H above: dFtcyDTDO (2.5 μM), Thapsigargin (400 nM), Tunicamycin (500 ng/ml), Cyclosporine A (10 μM), Dithiothreitol (DTT; (2.5 mM)), ISRIB (200 nM), or PERKi (1μM). O and M represent Oligomeric and Monomeric protein isoforms in panels A, C, E, G, and H.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Plasmid Preparation, Western Blot, Standard Deviation, Control, Knock-Out, Inhibition

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Genetic disruption of multiple DR5 disulfide bonds induces its stabilization and pro-apoptotic signaling. A. Structural model of DR5 showing its disulfide bonds, and the positive patch autoinhibitory domain described in the literature. B. Non-reducing immunoblot analysis of MDA-MB-468 cells engineered with doxycycline-inducible expression of wild type (WT) DR5 or the indicated Cys to Ser disulfide bond mutants. Cells were treated as indicated for 24 h with 1 μg/ml doxycycline and 2.5 μM dMtcyDTDO. The red arrow denotes DR4 oligomers that coincide with DR5 oligomerization. C. Reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 10 μM Q-VD-OPH. The catalog numbers of DR5 and DR4 antibodies are shown. D. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type DR4 and DR5, and DR4 and DR5 C-terminal deletion constructs defective in apoptotic signaling. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. E. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. F. Non-reducing immunoblot analysis of the indicated MDA-MB-468 doxycycline-inducible stable cell lines. Cells were treated for 24 h as indicated. G. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. O and M represent Oligomeric and Monomeric protein isoforms in panels B and D-G.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Disruption, Western Blot, Expressing, Stable Transfection, Construct

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: DDAs upregulate TRAIL Decoy Receptor 2, an effect overridden by Cyclosporine A. A. Sequence alignment of the putative autoinhibitory motifs of DR4, DR5, DCR1, and DCR2. B. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle, 5 μM Cyclosporine A or 100 nM FK506. C. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. D. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. Data are plotted as the average (N = 3), with error bars representing standard error. Asterisks denote p < 0.05 compared to control using Student’s unpaired t -test. E. Densitometry analysis of the relative levels of total, monomeric, and oligomeric forms of DR5 (left panel) or total levels of Cyclophilin B or DCR2 (right panel) from panels 7B-D. O and M represent Oligomeric and Monomeric protein isoforms in panels B-D.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Sequencing, Western Blot, Control

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Effects of altered disulfide bonding on DR5 cell surface localization and antibody recognition. A. Flow cytometry analysis of the indicated doxycycline-inducible MDA-MB-468 stable cell lines with an antibody to DR5 (Clone DJR2-4 (7-8)) (top panel) or DR4 (bottom panel). Prior to analysis, cells were treated for 24 h as indicated with 10 μM Q-VD-OPH, 1 μg/ml doxycycline, or 2.5 μM dFtcyDTDO. Dots represent the average values from three independent biological replicates performed in triplicate. * Represents p < 0.05, **** represents p < 0.0001, and ns represents not significant (p > 0.05). B. Non-reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines treated for 24 h as specified. Note the alternate staining patterns observed with different DR5 antibodies. O and M represent Oligomeric and Monomeric protein isoforms. C. The indicated MDA-MB-468 stable cell lines were treated for 24 h as indicated with 1 μg/ml doxycycline and 2.5 μM dFtcyDTDO and subjected to cell surface protein biotin labeling. Cell surface proteins (External; Ext.) were affinity purified with Streptavidin-agarose, and the unlabeled flow-through (Internal; Int.) proteins were also collected. Both fractions were analyzed by non-reducing immunoblot using the indicated antibodies. D. Cell surface protein labeling experiment as in panel C except that cell lines were treated with the indicated combinations of 1 μg/ml doxycycline, 2.5 μM dFtcyDTDO, and 10 μM Cyclosporine A.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Flow Cytometry, Stable Transfection, Western Blot, Staining, Labeling, Affinity Purification

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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 TRAIL‐R1 (DR4) (clone HS101) , AdipoGen , Cat#AG‐20B‐0022PF; RRID:AB_2490215.

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

Journal: Molecules

Article Title: Apoptosis Induced by (−)-Epicatechin in Human Breast Cancer Cells is Mediated by Reactive Oxygen Species

doi: 10.3390/molecules25051020

Figure Lengend Snippet: Schematic representation of (−)-epicatechin anticancer activity in MDA-MB-231 and MCF-7 cells. The anticancer effect in MDA-MB-231 cells was possibly triggered through TRAIL receptor interaction (DR4/DR5) and its upregulation. This extrinsic pathway activation was enhanced through the intrinsic pathway, resulting in caspases-dependent apoptosis and enhanced by the modulating of inhibitors of apoptosis by Smac/Diablo and HtrA2/Omi (continuous arrow). In MCF-7 cells, an anticancer effect was evident through the interaction with another receptor, the modulation of cellular kinases, and the upregulation of pro-apoptotic proteins such as Bad and Bax. This Bad and Bax upregulation induced the leak of cytochrome C, Smac/Diablo, and HtrA2/Omi into the cytoplasm, activating apoptosis through the intrinsic pathway. These mechanisms are tightly related to ROS generation (dashes arrow). (Arrows indicate activation, ⊥ indicates inhibition).

Article Snippet: As shown in , in MDA-MB-231 cells treated with (−)-epicatechin’s IC 50 value concentration, an increase was generated in the expression of the proteins as follows (compare A with B): TRAIL receptors (R1/DR4, R2/DR5) (C1–C2, C3–C4), pro-caspase 3 (line B9–B10), SMAC/Diablo (line D15–D16), and HTRA2/Omi (lines C21–C22).

Techniques: Activity Assay, Activation Assay, Inhibition