caspase 8 1c12  (Cell Signaling Technology Inc)

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: BMC Molecular and Cell Biology

Article Title: Alcohol exposure induces ferroptosis-dominated programmed cell death in esophageal epithelial cells

doi: 10.1186/s12860-026-00589-5

Figure Lengend Snippet: Long-term exposure to 5.0% ethanol triggers apoptosis, which undermines cell growth. A . An equal number of HEEC cells was seeded in 6-well plates and incubated with the complete medium containing DMSO (control), the medium containing 5.0% ethanol (EtOH), the medium containing 2 µM Z-LEHD-FMK (LEHD), or the medium containing 5.0% ethanol plus 2 µM LEHD for 30 min. Protein was extracted and analyzed by Western blotting. The membranes were probed for CASP8, CASP9, CASP3, or GAPDH. B . Quantitative analyses of CASP8, CASP9, and CASP3 expression against GAPDH in HEEC cells. * indicates a significant change compared to the control (DMSO). C . An equal number of HEEC cells was seeded on the coverslips and incubated with the complete medium (control) or the medium containing 5.0% ethanol for 5–30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for endoG. An FITC-conjugated secondary antibody was used to develop the signal. Cell nuclei were counterstained with DAPI. Scale bar = 5 μm. D . Quantitative analyses of endoG nuclear translocation in HEEC cells (%). * indicates a significant change compared to the control. E . An equal number of HEEC cells was seeded in 96-well plates and incubated with the medium containing 5.0% ethanol (EtOH) or the medium containing 5.0% ethanol plus 2 µM LEHD for 5 min, 30 min, 2 h, 6 h, or 12 h. Cell viability was assessed using CCK-8 kits. * indicates a significant change between EtOH and EtOH/LEHD. F . An equal number of Het1A cells was seeded in 96-well plates and incubated with the medium containing 5.0% ethanol (EtOH) or the medium containing 5.0% ethanol plus 2 µM LEHD for 5 min, 30 min, 2 h, 6 h, or 12 h. Cell viability was assessed using CCK-8 kits. * indicates a significant change between EtOH and EtOH/LEHD

Article Snippet: The following primary antibodies were used: Beclin-1 (Origene, #TA502643), PCNA, CASP1 (Abcam, #ab179515), CASP3 (Santa Cruz Biotechnology, #sc-271759), CASP8 (Origene, #TA374288), CASP9 (Origene, #TA4227045), endoG, GPX4 (Origene, #TA423164M), GSDMD, IL-1β (Santa Cruz Biotechnology, #sc-12742), MLKL, phosphor-MLKL (Abcam, #ab196436), SLC7A11 (Origene, #TA423232), LC3B, BAX, GAPDH (OriGene, #TA800894), and β-actin (Santa Cruz Biotechnology, Inc. #sc-69879).

Techniques: Incubation, Control, Western Blot, Expressing, Staining, Translocation Assay, CCK-8 Assay