Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 1 Involvement of Bid (BH3‐interacting domain death agonist) in the enhancement of cytotoxicity during treatment of artesunate (ART) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). HCT116 wild‐type (WT) and Bid knockout (KO) cells were pretreated with artesunate (ART, 50 μM) for 20 h and then exposed to human recombinant TRAIL (2ng/ml) for an additional 4 h. (a) Phase‐contrast images were visualized under a bright‐field microscope. Representative images are shown (scale bar: 100 µm). (b) Cell death was determined using a trypan blue exclusion assay. Error bars represent the mean± SD from triplicate experiments. For statistical analysis, two‐way analsis of variance followed by Tukey's post hoc test was used. ***p < 0.001. (c) For fluorescence images, cells were stained with propidium iodide (PI) and Hoechst 33342. Representative images are shown (scale bar: 25 µm).

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Recombinant, Microscopy, Trypan Blue Exclusion Assay, Fluorescence, Staining

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 2 Role of Bid (BH3‐interacting domain death agonist) in the synergistic interaction between artesunate/erastin (ART/ERA) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in HCT116 cells. HCT116 wild‐type (WT) and Bid knockout (KO) cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (a) Whole‐cell extracts were analyzed with an immunoblotting assay using indicated antibodies. (b) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance (ANOVA), followed by Tukey's post hoc test was used. ***p < 0.001 versus the Bid KO group. HCT116 WT cells were pretreated with ERA (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (c) Whole‐cell extracts were analyzed with the immunoblotting assay using indicated antibodies. (d) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. ***p < 0.001 versus the MOCK group. (e) Alteration of mitochondrial membrane potential was detected using JC‐1 dye after treatment. Red and green fluorescence represents the aggregate and monomeric form of JC‐1, respectively (scale bar: 50 μm). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Recombinant, Western Blot, Membrane, Fluorescence

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 4 Role of caspase‐8 in the synergistic interaction between artesunate (ART) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). HCT116 wild‐type (WT) cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. Caspase‐8 inhibitor Z‐IETD‐FMK (20 μM) was added 2 h before TRAIL treatment. (a) Cell death was determined using a trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way analysis of variance (ANOVA) was used. ***p < 0.001. (b) Whole‐cell lysates were analyzed with an immunoblotting assay using indicated antibodies. (c) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. ***p < 0.001. (d) Caspase‐8 activity was measured as described in Materials and Methods section. All experiments were performed in triplicates and caspase‐8 activity was plotted as average relative fluorescence units (RFUs). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Recombinant, Trypan Blue Exclusion Assay, Western Blot, Activity Assay, Fluorescence

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 5 Employment of Bid (BH3‐interacting domain death agonist) mutants for investigating the role of Bid in the synergistic apoptosis. HCT116 wild‐type (WT) or Bid knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with artesunate (ART) (50 μM) for 20 h and then exposed to human recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (2 ng/ml) for an additional 4 h. (a) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance (ANOVA), followed by Tukey's post hoc test was used. ***p < 0.001. (b, d) Cell lysates were analyzed with an immunoblotting assay using indicated antibodies. (c, e) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. **p < 0.01, ***p < 0.001 versus the vector group in (c). ***p < 0.001 versus the D60E group, ###p < 0.001 versus the G94E group in (e). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Stable Transfection, Expressing, Control, Plasmid Preparation, Recombinant, Trypan Blue Exclusion Assay, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 6 Translocation of truncated Bid (BH3‐interacting domain death agonist) to mitochondria during combined treatment of artesunate (ART) and tumor necrosis factor‐related apoptosis‐ inducing ligand (TRAIL). HCT116 wild‐type (WT) or Bid knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. After treatments, mitochondrial and cytosol fractions were isolated as described in the Materials and Methods section. Each fraction was analyzed with an immunoblotting assay using indicated antibodies. tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Translocation Assay, Knock-Out, Stable Transfection, Expressing, Control, Plasmid Preparation, Recombinant, Isolation, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 7 Artesunate (ART)‐induced endoplasmic reticulum stress response and death receptor 5 (DR5) expression. HCT116 wild‐type (WT) or Bid (BH3‐interacting domain death agonist) knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (2 ng/ml) for an additional 4 h. Z‐IETD‐FMK (20 μM) was added 2 h before TRAIL treatment. (a–d) Cell lysates were analyzed with an immunoblotting assay using indicated antibodies. Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way analysis of variance was used. ***p < 0.001 versus the MOCK group.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Expressing, Knock-Out, Stable Transfection, Control, Plasmid Preparation, Recombinant, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 8 Truncation of Bid (BH3‐interacting domain death agonist) in CHOP (CCAAT‐enhancer‐binding protein homologous protein)‐ deficient cells. mouse embryonic fibroblast (MEF) wild‐type (WT) and CHOP knockout (KO) cells were pretreated with artesunate (ART) (50 μM) for 20 h and then exposed to mouse recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (100 ng/ml) for an additional 4 h. (a) Whole‐cell extracts were analyzed with an immunoblotting assay using indicated antibodies. (b) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance, followed by Tukey's post hoc test was used. ***p < 0.001 versus the CHOP KO group. PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Binding Assay, Knock-Out, Recombinant, Western Blot