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