caspase 8 inhibitor  (InvivoGen)

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: 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:

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) and ELISA quantification of IL-1β (b) and TNFα (c) secretion in BMDMs from WT, Nlrp3 −/− , and Aim2 −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (d) Time-course of fungal hyphal growth (length/mm²) in WT and Nlrp3 −/− BMDMs left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (e) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) and (f) ELISA quantification of IL-1β secretion in BMDMs from WT, and Asc −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (g) Immunoblot for caspase-1 in BMDMs treated with caspase-1 inhibitor (VX765), caspase-8 inhibitor (Z-IETD-FMK), or both (VX765+Z-IETD-FMK), under single and sequential infections. (h) IL-1β measurement by ELISA in BMDMs treated with caspase-8 inhibitor Z-IETD-FMK, caspase-1 inhibitor VX765, their combination, left uninfected (Med) or after infection with P.a (MOI 0.005 and 0.01), and/or A.f (MOI 5). (i) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) in BMDMs from WT and Gsdmd −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5) and (j) corresponding IL-1β quantification for Gsdmd −/− . (k) Quantification of cell death, measured as the number of propidium iodide-positive (PI + ) cells, in Gsdmd −/− BMDMs following infection with P.a (MOI 0.01) and/or A.f (MOI 5). Data are from at least three independent experiments and presented as mean ± SEM. Statistical significance determined by ANOVA; *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: To evaluate inflammasome activation, BMDMs were treated with various inhibitors at the time of A. fumigatus superinfection, NLRPC inhibitor MCC950 (10 μM final concentration) (inhibit-mcc, InvivoGen), caspase-1 and caspase-4 inhibitor VX-765 (40 μM final concentration) (inh-vx765i-1, InvivoGen), caspase-8 inhibitor Z-IETD-FMK (20 μM final concentration) (inhibit-ietd, InvivoGen).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Infection