cleaved caspase 8  (ABclonal Biotechnology)

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: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A The mRNA expression of Caspase-8 in the border zone of infracted hearts from Sham and MI mice. n = 7 biologically independent mice. P = 0.0131. Statistical test was by a two-tailed unpaired t test. B The protein expression of Caspase-8 and Caspase-8-p18 in the border zone of the infracted hearts from Sham and MI mice. n = 9 biologically independent mice. P < 0.0001. C The protein expression of Gsdmd and Gsdmd-N in the border zone of infracted hearts from Sham and MI mice. n = 9 biologically independent mice. P < 0.0001. B , C Statistical test Two-Way ANOVA with Tukey’s multiple comparison. D Cleavage of Gsdmd in NMVCs from Caspase-8 overexpression, n = 5 biologically independent group. P < 0.0001. Statistical test was by a two-tailed unpaired t test. A – D Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Expressing, Two Tailed Test, Comparison, Over Expression

Journal: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A Schematic diagram of AAV9-Caspase-8 in vivo study (Created in BioRender. Liling, G. (2026) https://BioRender.com/4byuier ). B Representative echocardiograms from transthoracic two-dimensionally directed M-mode echocardiography, indicating cardiac function changes, n = 12 biologically independent mice. P < 0.0001. Statistical test was by a two-tailed unpaired t test. C H&E staining for morphological and histopathological examination of the myocardium, left: overall heart, right: enlargement of the area of apex in heart (green arrow: infiltration of inflammatory cell; black arrow: gap between cardiomyocytes), n = 6 (biologically independent mice). Scale bars, 50 μm. D Electron microscopy analysis ultra-microstructure of the heart (yellow arrow: mitochondrial swelling; red arrow: membrane rupture), n = 6 (biologically independent mice). Scale bars, 2 μm. E ELISA assay kit measured the LDH levels in the mice serum, n = 6 biologically independent mice, P = 0.0009. Statistical test was by a two-tailed unpaired t test. F ELISA assay kit measured the IL-1β ( P = 0.0034) and IL-18 ( P = 0.0034) levels in the mice serum, n = 6 biologically independent mice, Statistical test was by Two-tailed unpaired t test. G The protein expression of pyroptosis-related factors, including Gsdmd-N ( P = 0.0044), IL-1β ( P < 0.0001) and IL-18 ( P < 0.0001) in the heart of mice treated with AAV9-NC and AAV9-Caspase-8, n = 6 biologically independent mice, Statistical test Two-Way ANOVA with Tukey’s multiple comparison. H Immunofluorescence staining for examining punctate aggregates of Gsdmd-N on the cardiomyocyte membranes, n = 6 biologically independent mice. Scale bars, 2 μm. DAPI (blue), α-actinin (red), Gsdmd-N (green), yellow arrow: Gsdmd-N accumulation. B , E , F , G Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: In Vivo, Two Tailed Test, Staining, Electron Microscopy, Membrane, Enzyme-linked Immunosorbent Assay, Expressing, Comparison, Immunofluorescence

Journal: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A Construction flowchart of Gsdmd transgenic knockout mice (Created in BioRender. Di, H. (2026) https://BioRender.com/vtzrbhj ). B Representative western blots and statistical analysis of Gsdmd-N expression in Gsdmd -CKO mice treated with AAV9-Caspase-8 or not, n = 4 biologically independent mice. P = 0.0058 for Gsdmd flox/flox + AAV9-Caspase-8 vs. Gsdmd flox/flox , P = 0.0003 for Gsdmd -CKO + AAV9-Caspase-8 vs. Gsdmd flox/flox + AAV9-Caspase-8. Statistical test One-Way ANOVA with Tukey’s multiple comparison. C Representative echocardiograms from transthoracic two-dimensionally directed M-mode echocardiography, indicating cardiac function changes, n = 4 biologically independent mice. EF ( P = 0.0035) and FS ( P = 0.0062) for Gsdmd flox/flox + AAV9-Caspase-8 vs. Gsdmd flox/flox ; EF ( P = 0.012) and FS ( P = 0.0229) for Gsdmd -CKO + AAV9-Caspase-8 vs. Gsdmd flox/flox + AAV9-Caspase-8. Statistical test One-Way ANOVA with Tukey’s multiple comparison. D ELISA assay kit measured the IL-1β and IL-18 levels of each group mice, n = 4 biologically independent mice. IL-1β ( P = 0.001) and IL-18 ( P = 0.0002) for Gsdmd flox/flox + AAV9-Caspase-8 vs. Gsdmd flox/flox ; IL-1β ( P < 0.0001) and IL-18 ( P = 0.0136) for Gsdmd -CKO + AAV9-Caspase-8 vs. Gsdmd flox/flox + AAV9-Caspase-8. Statistical test One-Way ANOVA with Tukey’s multiple comparison. E H&E staining for morphological and histopathological examination of the myocardium, n = 4 biologically independent mice. Scale bars, 50 μm. B – D Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Transgenic Assay, Knock-Out, Western Blot, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A Detailed distribution of CG sites on Caspase-8 gene (Location of the CpG island) (Created in BioRender. Di, H. (2026) https://BioRender.com/3zy6o51 ). B Average methylation level of the CpG island of the Caspase-8 gene in mouse heart with MI or not, n = 3 biologically independent mice. For box plot, the center line represents the median (Sham: 0.01167, MI: 0.005); the bounds of the box represent the 25th and 75th percentiles (Sham: 0.01117-0.01333, MI: 0.004667-0.006); the whiskers extend to the smallest and largest values within 1.5 × IQR (Sham: 0.01033-0.014, MI: 0.004-0.007); data points beyond the whiskers are outliers. C The methylation levels at individual loci of the Caspase-8 gene’s CpG island, n = 3 biologically independent mice. D The mRNA of Caspase-8 in NMVCs after treatment with 5aza for 12 h and 24 h, n = 6 biologically independent group. P = 0.0004 for 5aza-24h vs. DMSO. Statistical test One-Way ANOVA with Tukey’s multiple comparison. E The protein expression of Caspase-8 in NMVCs after treatment with 5aza for 12 h and 24 h. n = 4 biologically independent group. Caspase-8 ( P = 0.044) and Caspase-8-p18 ( P = 0.0227) for 5aza-24h vs. DMSO. Statistical test Two-Way ANOVA with Tukey’s multiple comparison. F The changes of DNA methyltransferase, including Dnmt1 ( P = 0.000654), Dnmt3a ( P < 0.0001) and Dnmt3b ( P = 0.003231) protein expression during healthy and ischemic hearts, n = 5 biologically independent mice. Statistical test was by a Multiple Two-tailed unpaired t test. G 2% agarose gel analysis of ChIP-PCR products, n = 3 biologically independent group. H , I The mRNA and protein expression of Caspase-8 in NMVCs transfected with an overexpression plasmid of Dnmt3a, n = 6 biologically independent group. H P = 0.0005, (I) P < 0.0001. K , L The mRNA and protein expression of Caspase-8 in NMVCs transfected with si-Dnmt3a, n = 6 biologically independent group. K P = 0.0252, ( L ) Caspase-8, P = 0.0126; Caspase-8-p18, P = 0.0195. H , K Statistical test was by a two-tailed unpaired t test. I , L Statistical test Two-Way ANOVA with Tukey’s multiple comparison. J , M Caspase-8 methylation was analyzed by MSP, a method sensitive to low levels of methylation, during NMVCs treatment with overexpression and knockdown of Dnmt3a, n = 3 biologically independent group. D – F , H , I , K , L Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Methylation, Comparison, Expressing, Two Tailed Test, Agarose Gel Electrophoresis, Transfection, Over Expression, Plasmid Preparation, Knockdown

Journal: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A The transcriptional level of Caspase-8 in mice treated with MI and AAV9-Dnmt3a or not, n = 8 biologically independent mice. B The protein level of Caspase-8 in mice treated with MI and AAV9-Dnmt3a or not, n = 6 biologically independent mice. C The protein expression of pyroptosis-related factors: Gsdmd-N, IL-1β and IL-18, n = 5 biologically independent mice. A P = 0.0002, ( B , Caspase-8, P = 0.0311; Caspase-8-p18, P < 0.0001) ( C , Gsdmd-N, P < 0.0001; IL-1β, P = 0.0183; IL-18, P = 0.0248) for MI vs. Sham; ( A ) P = 0.0283, ( B , Caspase-8, P = 0.0065; Caspase-8-p18, P = 0.0028) ( C , Gsdmd-N, P < 0.0001; IL-1β, P = 0.0298; IL-18, P = 0.0189) for MI + AAV9-Dnmt3a vs. MI + AAV9-NC. A , C Statistical test One-Way ANOVA with Tukey’s multiple comparison. B Statistical test Two-Way ANOVA with Tukey’s multiple comparison. D Immunofluorescence staining for examining aggregates of Gsdmd-N on the cardiomyocyte membranes, n = 6 biologically independent mice. Scale bars, 10 μm. DAPI (blue), α-actinin (red), Gsdmd-N (green), yellow arrow: Gsdmd-N accumulation. E The protein expression of Gsdmd-N in NMVCs treated with si- Dnmt3a under control or si- Caspase-8 add Nec-1 (10 μM), n = 3 biologically independent group. F LDH release in culture medium from NMVCs transfected with si- Dnmt3a and si- Caspase-8 individually or collectively, n = 6 biologically independent group. E P = 0.0058, (F) P = 0.0003 for si-Dnmt3a vs. si-NC; ns: no significant for si-Dnmt3a + si-Caspase-8 + Nec-1 vs. si-Caspase-8 + Nec-1. E , F Statistical test One-Way ANOVA with Tukey’s multiple comparison. G Representative images of Hoechst33342/PI staining in NMVCs transfected with si- DNMT3a and si- Caspase-8 individually or collectively, n = 6 biologically independent group. Scale bars, 50 μm. A – C , E , F Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Expressing, Comparison, Immunofluorescence, Staining, Control, Transfection

Journal: Nature Communications

Article Title: Loss of DNA methyltransferase 3a enhances Caspase-8 transcription and promotes cardiomyocyte pyroptosis during myocardial infarction

doi: 10.1038/s41467-026-71941-8

Figure Lengend Snippet: A The flow diagram of UCSC, JASPAR and methylation-sensitive SELEX online website and database, it predicted the TFs based on CpG region (Created in BioRender. Liling, G. (2026) https://BioRender.com/9iigjgv ). B – E ChIP analysis of associations of Tfec, Mycn, Hes1 and Zbtb33 with Caspase-8 CpG island in NMVCs treated with Dnmt3a knock down and overexpression, n = 3 biologically independent group. D P = 0.0404 for OE-Dnmt3a vs. NC; P = 0.0088 for si-Dnmt3a vs. NC. F The transcription of Caspase-8 in NMVCs transfected with Hes1 overexpression plasmid separately, and co-transfected with Hes1 and Dnmt3a overexpression plasmid or Dnmt3a siRNA, n = 5 biologically independent group. G The protein level of Caspase-8 in NMVCs transfected with Hes1 overexpression plasmid separately, and co-transfected with Hes1 and Dnmt3a overexpression plasmid or Dnmt3a siRNA, n = 5 biologically independent group. F P = 0.0001, ( G ) P = 0.028 for OE-Hes1 vs. NC. ( F ) P = 0.0213 for OE-Hes1 + OE-Dnmt3a vs. OE-Hes1. F P = 0.0378, ( G ) P = 0.0353 for OE-Hes1 + si-Dnmt3a vs. OE-Hes1. ( B – G ) Statistical test One-Way ANOVA with Tukey’s multiple comparison. B – G Data are presented as mean ± SEM.

Article Snippet: After 2 h of blocking with 10% normal goat serum, samples were incubated with mixed primary antibodies against Caspase-8 (1:100, A0215, ABclonal, USA), Gsdmd-N (1:100, ab215203, Abcam, USA), and α-actinin (1:200, A7811, Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Methylation, Knockdown, Over Expression, Transfection, Plasmid Preparation, Comparison