z ietd fmk  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: A novel prenylflavonoid with efficacy in cancer therapy by targeting ceramide-orchestrated signaling

doi: 10.3389/fphar.2026.1862457

Figure Lengend Snippet: GH01 induces apoptosis in B16F10 cells via death receptor and mitochondrial pathways. B16F10 cells were treated with GH01 along with the caspase-8 inhibitor (Z-IETD-FMK, IETD) or the caspase-9 inhibitor (Z-LEHD-FMK, LEHD). (A) Representative TUNEL staining images indicating apoptosis in B16F10 cells (green, TUNEL; blue, DAPI/nuclear; scale bars = 50 μm). Quantification of TUNEL fluorescence. Values are presented as the means ± SD (n = 3). (B) Representative immunofluorescence images of B16F10 cells with caspase-3 expression (red, caspase-3; blue, DAPI/nuclear; scale bar = 50 μm). Quantification of caspase-3 fluorescence. Values are presented as the means ± SD (n = 3). Statistical significance was determined as * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the 20-μM group. P -values were obtained by one-way ANOVA with multiple comparisons.

Article Snippet: Both cells were also subjected to pre-treatment with caspase-8-inhibitor Z-IETD-FMK (MCE, USA) and caspase-9-inhibitor Z-LEHD-FMK (MCE, USA) with and without GH01 administration.

Techniques: TUNEL Assay, Staining, Fluorescence, Immunofluorescence, Expressing, Control

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-targeted TNFR1-agonists induce TNF-like caspase-1, caspase-3, and caspase-8 activation decoupled from NF-κB signaling in MCF-7 cells (A–C) Caspase-1/3/8 activation of HER2-expressing MCF-7 cells by ICM11 derivatives compared to (rh)TNF. MCF-7 cells were stimulated with increasing compound concentrations for 72 h. Caspase activities were detected intracellularly with FAM-FLICA(R) Caspase 1 Assay Kit (Biomol), CaspaTag Caspase-3 In situ Assay Kit (Merck Millipore), and CaspaTag Caspase-8 In Situ Assay Kit (Merck Millipore). Caspase-1/3/8 activation was normalized to (rh) TNF. (D) NF-κB activation in MCF-7 cells triggered by ICM11 derivatives and (rh) TNF. MCF-7 cells were stimulated with increasing concentrations of ICMs and (rh) TNF for 40 min. NF-κB was stained intracellularly with AF488-labeled anti-NF-κB staining antibody (BD) after lysis, fixation, and permeabilization of cells. (E) Remaining relative cell death of MCF-7 cells after treatment with ICM11 derivatives or (rh)TNF in the presence or absence of caspase inhibitors. MCF-7 cells were incubated with caspase-1 inhibitor (InvivoGen), caspase-3 inhibitor (R&D Systems), caspase-8 inhibitor (InvivoGen), or pan-caspase inhibitor (InvivoGen) at 50 μM and a fixed (rh) TNF or ICM concentration of 5 nM for 72 h. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain and normalized to (rh) TNF signal. Mean values ±SEM of four independent experiments for each figure are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: We also set out to investigate the consequences of caspase inhibition on cell death induction, harnessing caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen), caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems), and caspase-8 inhibitor (Z-IETD-FMK, InvivoGen) as well as pan-caspase inhibitor (zVAD-FMK, InvivoGen).

Techniques: Activation Assay, Expressing, In Situ, Staining, Labeling, Lysis, Incubation, Concentration Assay, Fluorescence

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: Caspase-1/3/8 and NF-κB activity of bispecific bivalent (2 + 2) and multivalent (4 + 2) and (6 + 2) ICMs in MCF-7 cells

Article Snippet: We also set out to investigate the consequences of caspase inhibition on cell death induction, harnessing caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen), caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems), and caspase-8 inhibitor (Z-IETD-FMK, InvivoGen) as well as pan-caspase inhibitor (zVAD-FMK, InvivoGen).

Techniques: Activity Assay, Activation Assay

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: Remaining cell death induction of MCF-7 cells after treatment with ICMs in the presence of different caspase inhibitors

Article Snippet: We also set out to investigate the consequences of caspase inhibition on cell death induction, harnessing caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen), caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems), and caspase-8 inhibitor (Z-IETD-FMK, InvivoGen) as well as pan-caspase inhibitor (zVAD-FMK, InvivoGen).

Techniques: Inhibition

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet:

Article Snippet: We also set out to investigate the consequences of caspase inhibition on cell death induction, harnessing caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen), caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems), and caspase-8 inhibitor (Z-IETD-FMK, InvivoGen) as well as pan-caspase inhibitor (zVAD-FMK, InvivoGen).

Techniques: Labeling, Isolation, Recombinant, Yeast, Expressing, Modification, Saline, Antibody Purification, Chromatography, Dip-and-Read Format, Staining, In Situ, cDNA Synthesis, Plasmid Preparation, Software, Analysis

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-targeted TNFR1-agonists induce TNF-like caspase-1, caspase-3, and caspase-8 activation decoupled from NF-κB signaling in MCF-7 cells (A–C) Caspase-1/3/8 activation of HER2-expressing MCF-7 cells by ICM11 derivatives compared to (rh)TNF. MCF-7 cells were stimulated with increasing compound concentrations for 72 h. Caspase activities were detected intracellularly with FAM-FLICA(R) Caspase 1 Assay Kit (Biomol), CaspaTag Caspase-3 In situ Assay Kit (Merck Millipore), and CaspaTag Caspase-8 In Situ Assay Kit (Merck Millipore). Caspase-1/3/8 activation was normalized to (rh) TNF. (D) NF-κB activation in MCF-7 cells triggered by ICM11 derivatives and (rh) TNF. MCF-7 cells were stimulated with increasing concentrations of ICMs and (rh) TNF for 40 min. NF-κB was stained intracellularly with AF488-labeled anti-NF-κB staining antibody (BD) after lysis, fixation, and permeabilization of cells. (E) Remaining relative cell death of MCF-7 cells after treatment with ICM11 derivatives or (rh)TNF in the presence or absence of caspase inhibitors. MCF-7 cells were incubated with caspase-1 inhibitor (InvivoGen), caspase-3 inhibitor (R&D Systems), caspase-8 inhibitor (InvivoGen), or pan-caspase inhibitor (InvivoGen) at 50 μM and a fixed (rh) TNF or ICM concentration of 5 nM for 72 h. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain and normalized to (rh) TNF signal. Mean values ±SEM of four independent experiments for each figure are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: To test the influence of the individual caspases (caspase-1, caspase-3 and caspase-8) on the killing of MCF-7 cells, TNF killing assay was performed with 5 nM ICM or TNF in presence of 50 μM caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen, inh-yvad) or 50 μM caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems, FMK004) or 50 μM caspase-8 inhibitor (Z-IETD-FMK, InvivoGen, inh-ietd) or pan-caspase inhibitor (zVAD-FMK, InvivoGen, tlrl-vad), respectively.

Techniques: Activation Assay, Expressing, In Situ, Staining, Labeling, Lysis, Incubation, Concentration Assay, Fluorescence

Journal: Genes to Cells

Article Title: Molecular Mechanism of Caspase‐8–Dependent Interleukin‐18 Activation in Pancreatic Cancer Cells Induced by 5‐Fluorouracil and Nutrient Starvation

doi: 10.1111/gtc.70111

Figure Lengend Snippet: Cleavage of IL‐18 and GSDMD by caspase‐8 activation in detached MIA PaCa‐2 cells induced by nutrient starvation and 5‐FU treatment. (A) Representative image of MIA PaCa‐2 cells treated with 5‐FU (25 μg/mL) for 48 h in low‐nutrient medium (×10). An enlarged view of the indicated region is shown; both attached and detached cells are present. (B) Experimental overview. Attached and detached fractions are denoted [A] and [D], respectively. (C) Attached and detached cells after 5‐FU treatment (25 μg/mL, 48 h) were collected separately and analyzed by western blotting with anti‐IL‐18 mAbs. β‐Actin was used as a loading control. (D) Lysates from (C) were analyzed by western blotting with each anti‐Caspase‐8 and anti‐GSDMD antibody. β‐Actin was used as a loading control. (E) Cells were pretreated with DMSO or the caspase‐8 inhibitor Z‐IETD‐FMK (20 μM) and then treated with 5‐FU (100 μg/mL) in low‐nutrient medium. After 48 h, detached cells were collected and analyzed by western blotting with the indicated antibodies. β‐Actin was used as a loading control.

Article Snippet: The caspase‐8 inhibitor Z‐IETD‐FMK was obtained from R&D Systems (USA).

Techniques: Activation Assay, Western Blot, Control

Journal: Genes to Cells

Article Title: Molecular Mechanism of Caspase‐8–Dependent Interleukin‐18 Activation in Pancreatic Cancer Cells Induced by 5‐Fluorouracil and Nutrient Starvation

doi: 10.1111/gtc.70111

Figure Lengend Snippet: Proposed model of pyroptosis induction in pancreatic cancer cells. Nutrient starvation induces caspase‐8–dependent cell pyroptosis accompanied by IL‐18 and GSDMD cleavage. 5‐FU increases the number of cells undergoing pyroptosis. Increased release of active IL‐18 can ultimately lead to chronic inflammation in the tumor environment.

Article Snippet: The caspase‐8 inhibitor Z‐IETD‐FMK was obtained from R&D Systems (USA).

Techniques: