agonistic anti-dr4  (Enzo Biochem)

Journal: bioRxiv

Article Title: Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via Death Receptor 4 Upregulation and Lipid Raft Localization

doi: 10.1101/2021.03.05.434100

Figure Lengend Snippet: (A-B) Confocal micrographs of HCT116 and SW620 cells, respectively. Red channel represents DR4, green is lipid rafts and blue is DAPI (nuclei). Scale bar = 30 m. (C) Quantification of DR4 area per cell in HCT116 and SW620 cells. For each cell line, N= μ 75 cells were analyzed. Data are presented as mean+SEM from N=3 independent experiments. ***p<0.001 ****p<0.0001 (unpaired two-tailed t-test). (D) OxR cells had increased surface expression of DR4 in non-permeabilized cells analyzed via flow cytometry. # Significant according to a chi-squared test (see Supplementary File 1). (E) Western blots for DR4 in whole cell lysates of parental and OxR cells. (F) Quantification of western blots from three independent experiments (N=3). Data are presented as mean+SEM. *p<0.05 (unpaired two-tailed t-test). (G) Percentage of apoptotic SW620 cells after treatment with 0.01-10 µg/ml Mapatumumab (sum of early and late-stage apoptotic cells from Annexin/PI staining). Data are presented as mean+SD. N=3 (n=9). ****p<0.0001 (multiple unpaired two-tailed t-tests). (H) Cell viability of SW620 cells after Mapatumumab treatment, determined by AnnexinV/PI staining. Data are presented as mean±SD. N=3 (n=6). (I) Maximum Mapatumumab sensitization within OxR cell lines compared to their parental counterparts. Data are presented as mean+SEM.

Article Snippet: Wells were treated in triplicate with soluble human TRAIL (PeproTech), or treated with the anti-DR4 agonist antibody Mapatumumab (Creative Biolabs, clone mAY4) and incubated for 24 hr.

Techniques: Two Tailed Test, Expressing, Flow Cytometry, Western Blot, Staining

Journal: bioRxiv

Article Title: Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via Death Receptor 4 Upregulation and Lipid Raft Localization

doi: 10.1101/2021.03.05.434100

Figure Lengend Snippet: (A-D) Confocal micrographs and DR5 quantification of HCT116, SW620, SW480 and HT29 cells, respectively. Red channel is death receptor 5, green is lipid rafts and blue is DAPI (nuclei). Scale bar = 30m. ** p<0.01 ****p<0.0001 (unpaired two-tailed t-test). Data are presented as mean+ SEM. For each cell line, N=75 cells were analyzed. (E ) OxR cells only demonstrate increased surface expression of DR5 in non-permeabilized SW620 cells. # Significant according to a chi-squared test (see Supplementary File 1).

Article Snippet: Wells were treated in triplicate with soluble human TRAIL (PeproTech), or treated with the anti-DR4 agonist antibody Mapatumumab (Creative Biolabs, clone mAY4) and incubated for 24 hr.

Techniques: Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via Death Receptor 4 Upregulation and Lipid Raft Localization

doi: 10.1101/2021.03.05.434100

Figure Lengend Snippet: (A) Cell viability of HCT116 cells after 0.01-10 µg/ml Mapatumumab treatment, determined by AnnexinV/PI staining. (B) Percentage of apoptotic SW620 cells after Mapatumumab treatment (sum of early and late-stage apoptotic cells from Annexin/PI staining). For all graphs, data are presented as mean+SD. N=3 (n=6). *p<0.05****p<0.0001 (multiple unpaired two-tailed t-tests).

Article Snippet: Wells were treated in triplicate with soluble human TRAIL (PeproTech), or treated with the anti-DR4 agonist antibody Mapatumumab (Creative Biolabs, clone mAY4) and incubated for 24 hr.

Techniques: Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

doi: 10.1038/cddis.2015.38

Figure Lengend Snippet: Withanolide E enhances TRAIL-induced extrinsic apoptotic pathway in ACHN cells. Cells were treated without (open bars) or with (filled bars) withanolide E for 3–4 h followed by agonistic anti-DR4 ( a ) or DR5 ( b ) antibodies for 20–24 h. For panels c – f , cells were treated 4 h±withanolide E (WE) then 4 h (for a total of 8 h) or 24 h±TRAIL in the continued presence of WE. Open bars: withanolide E only; black: withanolide E followed by TRAIL. ( c ) Cells were pretreated with ZVAD-FMK to block caspase activation followed by WE, then TRAIL. Caspase 8 ( d ) and caspase 3 ( e ) activities, and DNA fragmentation ( f ) were assessed using commercial kits. ( g ) Caspase cleavage and cFLIP degradation were assessed by immunoblot (total treatment time, 8 h). ( h ) TRAIL-dependent DISC formation was assessed in withanolide E- or bortezomib (Bzb – control with known activity)-treated ACHN cells (overnight treatment followed by biotinylated TRAIL at 4 °C, cell lysis, DISC precipitation and immunoblot). Error bars represent S.D. ( n =3)

Article Snippet: To assess death receptor utilization, agonistic anti-DR4 (Alexis/Enzo, Farmingdale, NY, USA) or DR5 (R&D Systems, Minneapolis, MN, USA) replaced TRAIL.

Techniques: Blocking Assay, Activation Assay, Western Blot, Activity Assay, Lysis

Journal: Cell Death & Disease

Article Title: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

doi: 10.1038/cddis.2015.38

Figure Lengend Snippet: Withanolide E effects on TRAIL sensitization mechanisms. ( a ) Effects of withanolide E on expression of pro- and antiapoptotic proteins. ACHN cells were treated 24 h with bortezomib (20 nM) or withanolide E, then±TRAIL (50 ng/ml) and immunoblot. ( b ) ACHN cells were treated±bortezomib (Bzb, positive control) or withanolide E (WE) followed by±TRAIL (total treatment time, 8 h) and immunoblot analysis of ER stress markers. ( c ) Cells were treated 4 h±withanolide E then 4 h (for a total of 8 h) or 24 h±TRAIL in the continued presence of WE. Open bars: withanolide E only; black: withanolide E followed by TRAIL. Mitochondrial potential was assessed (JC-1 assay). ( d ) Cells were treated with withanolide E (solid symbols) or withanolide A (inactive control, open symbols) and ROS generation estimated with DCFDA. ( e ) ACHN cells were pretreated with NAC (10 mM), Trolox (200 μ M) or vitamin C (vitC, 200 μ M) followed by withanolide E (2 h), then TRAIL (24 h). Open bars: withanolide E only; gray bars: TRAIL only; black bars: withanolide E+TRAIL. ( f ) ACHN cells were treated for 8 h or 24 h with 10 μ M withanolide E and assessed for expression of TRAIL receptors 1 and 2 by FACS (FACS Caliber, BD Biosciences). Antibodies used were (anti-TRAIL-R1 (human), mAb (HS101) (ATTO 488) or anti-TRAIL-R2 (human), mAb (HS201) (ATTO 647N) or mouse IgG1 atto488 or mouse IgG1atto 647 (Adipogen)). Dashed line represents isotype control, solid line DMSO-treated cells, and filled line withanolide E-treated cells. Error bars represent S.D. ( n =3–4)

Article Snippet: To assess death receptor utilization, agonistic anti-DR4 (Alexis/Enzo, Farmingdale, NY, USA) or DR5 (R&D Systems, Minneapolis, MN, USA) replaced TRAIL.

Techniques: Expressing, Western Blot, Positive Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: SPR measurements of DR4 and DR5 binding affinities and competitive ELISA for DR4 and DR5, and decoy receptors, DcR1 and DcR2. Apparent K A and EC 50 ratios were calculated relative to WT rhTRAIL.

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Binding Assay, Competitive ELISA

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: DR4-selectivity of rhTRAIL-C3. DR4-responsive EM-2 chronic myelogenous leukaemia cells (A) and DR5-responsive A2780 ovarian carcinoma cells (B) were treated with increasing doses of rhTRAIL-C3 (G131R/N199R/K201H), WT rhTRAIL or DR5-selective rhTRAIL variant (D269H/E195R) for 24 hrs. Induction of apoptosis was determined using annexin V assay. The graphs show the percentage of dead cells ± S.E.M. determined with flow cytometric analysis of annexin V positivity in three independent experiments.

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Variant Assay, Annexin V Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: Deficiency of DR4 blocks rhTRAIL-C3 induced apoptosis. WT BJAB cells (A), BJAB cells overexpressing GFP (B), non-functional, C-terminal deletion mutants of DR4 (C) or DR5 (D) were treated with WT rhTRAIL and rhTRAIL-C3. Induction of apoptosis was determined by measuring loss of mitochondrial membrane potential (ΔΨm).

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Functional Assay, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: Cell surface expression of TRAIL receptors in OCI-AML3, HL-60 and MOLM-13 cells. Cell surface expression of DR4, DR5, DcR1 and DcR2 was measured by immunostaining and detected with flow cytometry (FacsCanto II, Beckton Dickinson). The histograms are representatives of three independent experiments. Red filled peaks: isotype control, green open peaks: TRAIL receptor stained sample.

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Expressing, Immunostaining, Flow Cytometry, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: Functionality of DR4 and DR5 in AML cells. HL60 (A), ML-1 (B), MOLM-13 (C) OCI-AML3 (D) and EM-2 cells were treated with WT rhTRAIL, agonistic anti-DR4 and anti-DR5 antibodies for 24 hrs with or without previous crosslinking of the antibodies. Induction of cell death, as a measure of receptor functionality, was measured with 7-aminoactinomycin D staining and flow cytometric analysis. Graphs demonstrate the average percentage of dead cells (±S.E.M.) from three independent experiments.

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting AML through DR4 with a novel variant of rhTRAIL

doi: 10.1111/j.1582-4934.2010.01211.x

Figure Lengend Snippet: TRAIL receptor cell surface expression in primary AML blasts

Article Snippet: The functionality of DR4 and DR5 in HL-60, ML-1, MOLM-13, OCI-AML3 and EM-2 cells was determined using agonistic anti-DR4 and anti-DR5 antibodies (Novartis).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

doi: 10.1038/sj.bjc.6605021

Figure Lengend Snippet: TRAIL can activate the JNK pathway via both DR4 and DR5 in colon cancer cell lines. ( A ) Western blot analysis of total JNK and p-JNK in Colo205, HCT15 and HCA7 cell lysates following treatment with 20 ng ml −1 rhTRAIL for Colo205 cells and 50 ng ml −1 for HCT15 and HCA7 for the times indicated. Total JNK levels and actin were also detected as loading controls. ( B ) Western blot analysis of total c-Jun and p-c-Jun levels in Colo205, HCT15 and HCA7 cell lysates following rhTRAIL treatment as above. ( C ) Cell surface expression of DR4 and DR5 in Colo205 and HCT15 cells measured by immunostaining followed by flow cytometry. Each histogram shows an overlay of a negative control (2° AB), DR4 and DR5 receptors. ( D ) Apoptosis-inducing potential of DR4 and DR5 in Colo205 and HCT15 cells. Cells were treated with cross-linked agonistic DR4 or DR5 antibodies (5 n M for Colo205 and 10 n M for HCT15) or rhTRAIL (20 and 50 ng ml −1 for Colo205 and HCT15) for 3 h in Colo205 cells and for 5 h in HCT15 cells. Apoptosis induction was measured with Annexin V. The graphs show averaged percentage of apoptotic cells±s.e.m. of three independent experiments. ( E ) Western blot analysis showing activation of JNK via DR4 and DR5 in Colo205 and HCT15 cells. Cells were treated with agonistic DR4 and DR5 antibodies (5 n M for Colo205 cells and 10 n M for HCT15 cells) for the times indicated. JNK phosphorylation was detected in whole cell lysates. The images shown are representatives of three independent experiments. Actin was detected as a loading control.

Article Snippet: Cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114–281; Triskel Therapeutics, Groningen, The Netherlands), agonistic anti-DR4 or anti-DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland).

Techniques: Western Blot, Expressing, Immunostaining, Flow Cytometry, Negative Control, Activation Assay, Phospho-proteomics, Control

Journal: British Journal of Cancer

Article Title: Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

doi: 10.1038/sj.bjc.6605021

Figure Lengend Snippet: Inhibition of JNK inhibits rhTRAIL-mediated apoptosis, but potentiates DR4- or DR5-mediated apoptosis in colon cancer cells. ( A ) L -JNKI can inhibit rhTRAIL-mediated JNK activation. HCT15 cells were preincubated for 1 h with L -JNKI (25 μ M ) followed by treatment with 50 ng ml −1 rhTRAIL for 3 h or UV for 30 min. The figure shows GST p-c-Jun phosphorylation by JNK as a measure of JNK activity. ( B ) Inhibition of L -JNKI potentiates apoptosis induced by selective activation of DR4 and DR5, but reduces apoptosis induced by rhTRAIL. Colo205 cells were treated for 3 h with either 20 ng ml −1 rhTRAIL or 5 n M of DR4/DR5 agonistic antibodies with or without 30 min pretreatment with L -JNKI (50 μ M ) and induction of apoptosis was measured with Annexin V. The graph shows average cell death±s.e.m. of four independent experiments. The asterisk ( * ) indicates significant differences ( P <0.05). ( C ) Inhibition of L -JNKI potentiates apoptosis induced by selective activation of DR4 or DR5, but reduces apoptosis induced by rhTRAIL. HCT15 cells were treated for 3 h with either 50 ng ml −1 rhTRAIL or 10 n M of DR4/DR5 agonistic antibodies with or without 30 min pretreatment with L -JNKI (50 μ M ) and induction of apoptosis was measured with Annexin V. The graph shows average cell death±s.e.m. of three independent experiments. The asterisk ( * ) indicates significant differences ( P <0.05).

Article Snippet: Cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114–281; Triskel Therapeutics, Groningen, The Netherlands), agonistic anti-DR4 or anti-DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Activity Assay

Journal: British Journal of Cancer

Article Title: Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

doi: 10.1038/sj.bjc.6605021

Figure Lengend Snippet: Receptor internalisation induced by rhTRAIL and agonistic DR4/5 antibodies. ( A ) Flow cytometric analysis of ligated receptor internalisation in Colo205 cells. Cells were treated with DR4/5 agonistic antibodies (5 n M ) cross-linked with FITC-labelled secondary antibody or FITC-labelled rhTRAIL (20 ng ml −1 ) for 30 min at 37°C or at 4°C. Cells were washed with 0.2 M acetic acid to remove surface bound rhTRAIL and agonistic antibodies. ( B ) Quantification of receptor internalisation. The graph shows the difference of isotype control-normalised MFI measured at 4 and 37°C (normalised MFI at 37°C minus normalised MFI at + 4°C). The first set of bars shows the level of receptor internalisation occurred at 37°C after treatment with rhTRAIL or agonistic antibodies (normalised MFI at 37°C/acid wash – normalised MFI at + 4°C/acid wash). The second set of bars indicates that rhTRAIL and agonistic DR4- and DR5 antibodies bound to the TRAIL receptors to a similar level regardless of the incubation temperature. ( C ) Induction of apoptosis by treatments detailed in point a. After the 30 min incubation with rhTRAIL or antibodies, the unbound molecules were removed by a wash step and the cells were incubated in normal growth medium for an additional 3 h after which induction cell death was measured with Annexin V. The data shown are representative of three independent experiments.

Article Snippet: Cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114–281; Triskel Therapeutics, Groningen, The Netherlands), agonistic anti-DR4 or anti-DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland).

Techniques: Control, Incubation

Journal: British Journal of Cancer

Article Title: Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

doi: 10.1038/sj.bjc.6605021

Figure Lengend Snippet: rhTRAIL and selective activation of DR4 or DR5 leads to activation of distinct JNK1 isoforms. ( A ) Phosphorylation of JNK1 isoforms by rhTRAIL and agonistic DR4/5 antibodies. Colo205 cells were treated with 5 n M of agonistic DR4/5 antibodies or 20 ng ml −1 rhTRAIL. HCT15 cells treated with 10 n M of agonistic DR4/5 antibodies or 50 ng ml −1 rhTRAIL for 3 h. JNK1 was immunoprecipitated (IP) and its phosphorylation pattern was assessed by western blotting with p-JNK antibody. JNK1 western blots show total JNK1 protein immunoprecipitated from cell lysates that was used for quantification of the amount of p-JNKI in the IP. ( B ) Densitometric quantification of p-JNK1 levels. The graph shows averaged p-JNK1 band densities normalised for total JNK1 levels in the immunoprecipitates of Colo205 (top) and HCT15 (bottom) cells from four independent experiments. ( C ) Phosphorylation of JNK2 isoforms by rhTRAIL and agonistic DR4/5 antibodies. Colo205 and HCT15 cells were treated as in point A and p-JNK protein (both JNK1 and 2) was immunoprecipitated. JNK2 phosphorylation was assessed in the p-JNK immunoprecipitates by probing the blots with a JNK2-specific antibody. P-JNK protein levels were also determined for quantification of JNK2 phosphorylation. ( D ) Densitometric quantification of p-JNK2 levels in Colo205 (left) and HCT15 (right) cells. The graph shows averaged p-JNK2 band densities normalised for total JNK2 levels in the immunoprecipitates from two independent experiments.

Article Snippet: Cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114–281; Triskel Therapeutics, Groningen, The Netherlands), agonistic anti-DR4 or anti-DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland).

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Western Blot

Journal: British Journal of Cancer

Article Title: Differential activation of JNK1 isoforms by TRAIL receptors modulate apoptosis of colon cancer cell lines

doi: 10.1038/sj.bjc.6605021

Figure Lengend Snippet: JNK1 α 1 has an antiapoptotic function. ( A ) Knockdown of JNK1 α 1 in Colo205 cells by JNK1 α 1 shRNA expression plasmid. Cells were nucleofected with JNK1 α 1 or scrambled shRNA expression plasmids. Total RNA was isolated 24 h after transfection and JNK expression was detected with primers detecting all JNK1 mRNA isoforms or primers specific to JNK1 α 1 or JNK1 β 1 isoforms. ( B ) Western blot analysis of the expression of the short (JNK1 α 1 and β 1) and long (JNK1 α 2 and β 2) isoforms of JNK1 in JNK1 α 1 shRNA expressing cells. ( C ) Knockdown of JNK1 α 1 increases apoptosis induced by selective activation of DR4 or DR5. Colo205 cells were treated with agonistic DR4/5 antibodies (5 n M ) or rhTRAIL (20 ng ml −1 ) for 3 h, after which percentage of apoptotic cells was determined with Annexin V. The graph shows the average cell death induced±s.e.m. of five independent experiments (sum of Ann V + /PI − and Ann V + /PI + percentages). The * indicates significant differences ( P <0.05).

Article Snippet: Cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114–281; Triskel Therapeutics, Groningen, The Netherlands), agonistic anti-DR4 or anti-DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland).

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Isolation, Transfection, Western Blot, Activation Assay