z devd fmk  (Santa Cruz Biotechnology)

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: Journal of Advanced Research

Article Title: Designing an apoptosis reporter by mutagenesis-based insertion of caspase-3 cleavage motif into green fluorescence protein

doi: 10.1016/j.jare.2025.06.070

Figure Lengend Snippet: Monitoring of apoptosis reporter in live cells. (A) HEK293 EGFP #4-8 cells were cultured with or without 1 μM STA, and images were obtained every 30 min for 48 h; scale bar 50 μm. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). (B) Protein levels of GFP, caspase-3, cleaved caspase-3, cytochrome C , PARP, and GAPDH by Western blotting in HEK293 EGFP #4-8 cells incubated with STA. Graph, protein levels (N = 3). (C) HEK293 EGFP #4-8 cells were cultured with 1 μM STA co-treated with or without 20 μM Z-DEVD-FMK and 10 μM Z-YVAD-FMK, and images were obtained every 30 min for 48 h. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). (D) HEK293 EGFP #4-8 cells were cultured with 100, 200, and 500 μM H 2 O 2 , and images were obtained every 30 min for 24 h. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). Error bars are ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Article Snippet: Z-DEVD-FMK (caspase-3 inhibitor, #sc-311558) and Z-YVAD-FMK (caspase-1 inhibitor, #sc-3071) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Fluorescence, Software, Western Blot, Incubation