Journal: BMC Immunology

Article Title: Establishment and validation of a recurrent prediction model for glioma: extrinsic apoptotic molecules FADD and CASP8 are closely associated with glioma recurrence

doi: 10.1186/s12865-025-00746-z

Figure Lengend Snippet: The association between recurrent score and classical apoptotic genes. (A) The relationship between the 6 genes and recurrent score in CGGA and TCGA database . (B) PPI network of CASP3, CASP9, FADD, CASP7, CASP8, BCL2，and the 9-gene signature from the STRING. (C-D) The expression levels of the 6 apoptotic genes in low- and high-risk levels . (E-J) Correlation between recurrent score and expression levels of apoptotic genes. *P<0.05; ***P<0.001; ns, not significant

Article Snippet: Characterizing the differential expression patterns of CASP8 and FADD in gliomas and normal tissues will play a crucial role in the further development of targeted therapeutic strategies for gliomas Fig. 9 RNA and protein levels of CASP8 and FADD in normal tissues and tumors. (A-B) RNA expression of CASP8 and FADD in normal tissues from the NCBI database (https://www.ncbi.nlm.nih.gov/). (C-D) RNA expression of CASP8 and FADD in normal tissues from the Human Protein Atlas database (https://www.proteinatlas.org/). (E) Protein levels of CASP8 and FADD in normal brain tissues from The Human Protein Atlas database. (F) Protein levels of CASP8 and FADD in normal tissues from The Human Protein Atlas database. (G) Protein levels of CASP8 and FADD in tumors from The Human Protein Atlas database

Techniques: Expressing

Journal: BMC Immunology

Article Title: Establishment and validation of a recurrent prediction model for glioma: extrinsic apoptotic molecules FADD and CASP8 are closely associated with glioma recurrence

doi: 10.1186/s12865-025-00746-z

Figure Lengend Snippet: RNA and protein levels of CASP8 and FADD in normal tissues and tumors. (A-B) RNA expression of CASP8 and FADD in normal tissues from the NCBI database (https://www.ncbi.nlm.nih.gov/). (C-D) RNA expression of CASP8 and FADD in normal tissues from the Human Protein Atlas database (https://www.proteinatlas.org/). (E) Protein levels of CASP8 and FADD in normal brain tissues from The Human Protein Atlas database. (F) Protein levels of CASP8 and FADD in normal tissues from The Human Protein Atlas database. (G) Protein levels of CASP8 and FADD in tumors from The Human Protein Atlas database

Article Snippet: Characterizing the differential expression patterns of CASP8 and FADD in gliomas and normal tissues will play a crucial role in the further development of targeted therapeutic strategies for gliomas Fig. 9 RNA and protein levels of CASP8 and FADD in normal tissues and tumors. (A-B) RNA expression of CASP8 and FADD in normal tissues from the NCBI database (https://www.ncbi.nlm.nih.gov/). (C-D) RNA expression of CASP8 and FADD in normal tissues from the Human Protein Atlas database (https://www.proteinatlas.org/). (E) Protein levels of CASP8 and FADD in normal brain tissues from The Human Protein Atlas database. (F) Protein levels of CASP8 and FADD in normal tissues from The Human Protein Atlas database. (G) Protein levels of CASP8 and FADD in tumors from The Human Protein Atlas database

Techniques: RNA Expression

Journal: Journal of Biological Chemistry

Article Title: Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5

doi: 10.1074/jbc.m401056200

Figure Lengend Snippet: FIG. 3. DR5 and c-FLIPL interact in cell culture. A and B, interaction of c-FLIPL and DR5 proteins in co-immunoprecipitation studies. HEK 293 cells were co-transfected with expression vectors for vector alone, FLAG-tagged c-FLIPL, DR5, or c-FLIPL and DR5 as indicated. FLAG immunoprecipitates were analyzed for the presence of DR5 and DR5 immunoprecipitates were analyzed for the presence of c-FLIPL. The presence of DR5 and c-FLIP in cell extracts were verified by immunoblotting for DR5 or c-FLIP. C, interaction between c-FLIPp12 and DR5 in co- immunoprecipitation studies. HEK 293 cells were co-transfected with expression vectors for vector alone, FLAG-tagged c-FLIPp12, DR5, or c-FLIP12 and DR5 as indicated. FLAG immunoprecipitates were analyzed for the presence of DR5 and DR5 immunoprecipitates were analyzed for the presence of c-FLIPp12. The presence of DR5 in total cell lysate is shown by Western blot analysis with an anti-Myc antibody. D, interaction between endogenous c-FLIPL and endogenous DR5 is independent of FADD. Association of endogenous DR5 with endogenous c-FLIPL in parental Jurkat A3 and FADD-deficient Jurkat A3 cells is shown by immunoprecipitation with DR5 antibody followed by immunoblotting with c-FLIP antibody. The level of DR5 protein in the immunocomplex is determined by immunoblotting with DR5 antibody. An unrelated immune serum is used as a negative control. In the bottom panel, the level of FADD expression is shown for each cell line.

Article Snippet: The following antibodies were used in immunocomplex and DISC analysis: caspase-8 1C12 (Cell Signaling Technology, Beverly, MA), caspase-10 N-19 (Santa Cruz Biotechnology, Santa Cruz, CA), FADD H181 (Santa Cruz Biotechnology), FLIP AF821 (R&D Systems, Minneapolis, MN), FLIP G11 (Santa Cruz Biotechnology), and FLIP Dave 2 (Alexis Corporation).

Techniques: Cell Culture, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5

doi: 10.1074/jbc.m401056200

Figure Lengend Snippet: FIG. 4. c-FLIPL interacts with DR5 in the absence of TRAIL stimulation and upon formation of the FADD/caspase-8 DISC, DR5, and c-FLIPL interaction is diminished. Time course of c- FLIPL and DR5 interaction and FADD/caspase-8 DISC formation was carried out in Jurkat cells that were treated with 10 ng/ml of TRAIL for 0, 5, 15, 30, 60, and 120 min. In stimulated cells, upon TRAIL treatment (10 ng/ml) for 1 h, cells were lysed and the assembled DISCs were immunoprecipitated with protein A and analyzed by Western blotting using antibodies to FADD, caspase-8, caspase-10, TRADD, c-FLIPL, c-FLIPp43, and c-FLIPs In unstimulated condition, cells were first lysed, and then TRAIL was added followed by analysis of the immunocomplex. In the absence of TRAIL treatment, c-FLIPL is associated with TRAIL receptor. Approximately, 5 min after TRAIL treatment FADD and caspase-8 are recruited to the DISC. At the same time, the level of c-FLIPL is diminished and the levels of c-FLIPp43 and c-FLIPS are increased in the complex.

Article Snippet: The following antibodies were used in immunocomplex and DISC analysis: caspase-8 1C12 (Cell Signaling Technology, Beverly, MA), caspase-10 N-19 (Santa Cruz Biotechnology, Santa Cruz, CA), FADD H181 (Santa Cruz Biotechnology), FLIP AF821 (R&D Systems, Minneapolis, MN), FLIP G11 (Santa Cruz Biotechnology), and FLIP Dave 2 (Alexis Corporation).

Techniques: Immunoprecipitation, Western Blot

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency disrupts PANoptosome assembly and augments RIPK1/3‐ dependent necrosome formation in vivo. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 8 h post‐CLP. Co‐immunoprecipitates were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images of lung macrophages stained for CD68 (green), Caspase‑8 (red), and ASC (cyan). Merged images indicate ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 50 µm. Statistical significance was determined by using one‑way ANOVA with Bonferroni's post hoc test; * p < .05, ** p < .01 and ns indicates p > .05.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: In Vivo, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency impairs PANoptosome assembly and enhances RIPK1/3‐dependent necrosome formation in macrophages. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 3 h post‐LPS. Co‐IP were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images showing the co‑localisation of RIPK3 (cyan), ASC (green), and Caspase‑8 (red) in PBS‐ or LPS‐treated BMDMs. Merged images indicate RIPK3–ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 25 µm (merged), 10 µm (zoomed). Statistical differences were analysed by one‑way ANOVA with Bonferroni's post hoc test, * p < .05 and ** p < .01.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Western Blot, Isolation, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Pharmaceuticals

Article Title: Carboxyl Group-Modified Myoglobin Induces TNF-α-Mediated Apoptosis in Leukemia Cells

doi: 10.3390/ph15091066

Figure Lengend Snippet: SEM-Mb-treated U937 cells underwent apoptosis. Without specific indication, U937 cells were treated with 1.5 μM SEM-Mb for 24 h. ( A ) SEM-Mb induced cell death in a concentration- and time-dependent manner. U937 cells were incubated with varying concentrations of SEM-Mb or Mb for 24 h. Cell viability was determined by MTT assay. Results are expressed as the percentage of cell survival relative to the control. Each value is the mean ± SD of triplicate determinations. (Inset) U937 cells were incubated with 1.5 μM SEM-Mb for indicated time periods. ( B ) Flow cytometry analyses of SEM-Mb-treated U937 cells using annexin V-FITC/propidium iodide double staining. (Left) Untreated control cells. (Right) U937 cells were treated with 1.5 μM SEM-Mb for 24 h. On flow cytometric scatter graphs, the left lower quadrant represents remaining live cells. The right lower quadrant represents the population of early apoptotic cells. The right upper quadrant represents the accumulation of late apoptotic cells. ( C ) Western blot analyses showing the degradation of procaspase-3/-8 and PARP in SEM-Mb-treated cells. ( D ) Pretreatment with caspase inhibitors restored the viability of SEM-Mb -treated cells. U937 cells were pretreated with 10 μM Z-DEVD-FMK (caspase-3 inhibitor) or Z-IETD-FMK (caspase-8 inhibitor) for 1 h, and then incubated with 1.5 μM SEM-Mb for 24 h. Cell viability was determined by MTT assay. The values represent averages of three independent experiments with triplicate measurement (mean ± SD, * p < 0.05). ( E ) Effect of SEM-Mb on the production of t-Bid and the expression of BCL2 family proteins. ( F ) Dissipation of mitochondrial membrane potential (∆Ψm) in SEM-Mb-treated cells. U937 cells were treated with 1.5 μM SEM-Mb for 24 h, and then incubated with rhodamine 123 for 15 min. The loss of ∆Ψm was analyzed by flow cytometry. ( G ) Transfection of FADD siRNA inhibited SEM-Mb-induced the degradation of procaspase-8/-3 and the production of t-Bid. U937 cells were transfected with 100 nM control siRNA or FADD siRNA, respectively. After 24 h post-transfection, the cells were treated with 1.5 μM SEM-Mb for 24 h. ( H ) Depletion of FADD inhibited SEM-Mb-induced cell death (mean ± SD, * p < 0.05).

Article Snippet: Gene silencing experiments were performed using FADD siRNA, NOX4 siRNA, and negative control siRNA (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Incubation, MTT Assay, Control, Flow Cytometry, Double Staining, Western Blot, Expressing, Membrane, Transfection

Journal: Pharmaceuticals

Article Title: Carboxyl Group-Modified Myoglobin Induces TNF-α-Mediated Apoptosis in Leukemia Cells

doi: 10.3390/ph15091066

Figure Lengend Snippet: NOX4-mediated SIRT3 degradation regulated p38 MAPK/TTP axis-dependent TNF-α upregulation in SEM-Mb-treated cells. U937 cells were directly treated with 1.5 μM SEM-Mb for 24 h or pre-treated with 1 μM MG132 or 10 μM GLX351322 for 1 h and then incubated with 1.5 μM SEM-Mb for 24 h. ( A ) Effect of SEM-Mb on SIRT3 expression. ( B ) qRT-PCR analyses of SIRT3 mRNA levels in SEM-Mb-treated cells (mean ± SD, NS, not statistically significant). Effect of ( C ) MG132 and ( D ) GLX351322 on the SIRT3 expression in SEM-Mb-treated cells. ( E ) Effect of NOX4 depletion on SIRT3 expression in U937 cells. U937 cells were transfected with 100 nM negative control siRNA or NOX4 siRNA, respectively. After 24 h post-transfection, the cells were treated with 1.5 μM SEM-Mb for 24 h. ( F ) Effect of SIRT3 overexpression on SEM-Mb-induced the production of mitochondrial ROS in U937 cells (mean ± SD, * p < 0.05). U937 cells were transfected with empty vector or pCMV3-N-His-SIRT3, respectively. After 24 h post-transfection, the transfected cells were treated with 1.5 μM SEM-Mb for 24 h. ( G ) Effect of SIRT3 overexpression on the viability of SEM-Mb-treated cells (mean ± SD, * p < 0.05). ( H ) Effect of SIRT3 overexpression on the expression of p-p38 MAPK, TTP, and TNF-α in SEM-Mb-treated U937 cells.

Article Snippet: Gene silencing experiments were performed using FADD siRNA, NOX4 siRNA, and negative control siRNA (Santa Cruz Biotechnology).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Over Expression, Plasmid Preparation

Journal: Nature Communications

Article Title: Prussian blue nanoparticles targeting multiple PANoptosome-mediated PANoptosis for myocardial ischemia-reperfusion injury therapy

doi: 10.1038/s41467-026-70012-2

Figure Lengend Snippet: A Schematic of ischemic zone (IZ), remote zone (RZ), and border zone (BZ) in human acute myocardial infarction. The heart element in the image was sourced from BioRender (Created in BioRender. xu, L. (2026) https://BioRender.com/9kka4p3 ). B UMAP visualization of cell distribution (left) and annotated cell types (right) in the control ( n = 4) versus IZ ( n = 11) groups. C Cardiomyocyte subpopulation quantification (left) and UMAP-based clustering (right) in the control and IZ groups. D , E UMAP projection ( D ) and box plots ( E ) showing PANoptosis activation across cardiac cell types. (The exact n in E = [left to right] 20, 84 cells; 17326, 3506 cells; 6231, 6969 cells; 9468, 12187 cells; 3386, 7142 cells; 3039, 2286 cells; 583, 254 cells; 531, 1138 cells). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right: 2.39E-265, 1.55E-300, 1.05E-35, 4.09E-05, 2.21E-06, 0.0002, 1.9E-11. F Violin plots comparing PANoptosis-related gene expression ( AIM2, ZBP1, RIPK1, Pyrin, NLRP12, NLRP3, MLKL, GSDMD, RIPK3, caspase 3, caspase 1 , and caspase 8 ) in total cells. ( n = 40706 cells [control], 33688 cells [IZ]). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right ( AIM2 to caspase 8 ): 0.2752, 0.0002, 0, 0.0854, 6.67E-09, 7.19E-82, 3.59E-37, 0.0709, 0.0269, 2.29E-280, 2.45E-30, 4.07E-09. G, H Cardiomyocyte-specific UMAP ( G ) and quantitative PANoptosis levels ( H ) of the control and IZ groups. (The exact n in H = [left to right] 6904 cells, 0; 5281 cells, 0; 1191, 3346 cells; 3192, 80 cells; 758, 80 cells). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right: 4.01E-05, 0.0021. I Proportional representation of cardiomyocyte subpopulations. J , K Heatmap depicting activation patterns of apoptosis/PANoptosis/pyroptosis/necroptosis ( J ) and PANoptosis-related genes ( K ) across cardiomyocyte subtypes. Significance: wilcox.test, a two-sided test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns : no significant difference ( P > 0.05). Source data are provided as a Source Data file.

Article Snippet: CD41 (24552-1-AP), Collagen III (22734-1-AP), AIM2 (20590-1-AP), caspase 1 (81482-1-RR), and caspase 8 (13423-1-AP) antibodies were bought from Proteintech, China.

Techniques: Control, Activation Assay, Gene Expression

Journal: Cell death & disease

Article Title: c-FLIP and CD95 signaling are essential for survival of renal cell carcinoma.

doi: 10.1038/s41419-019-1609-y

Figure Lengend Snippet: Fig. 1 Cycloheximide sensitizes clearCa cells towards CD95L-induced apoptosis. a Surface expression of death receptors CD95, TRAIL-R1, TRAIL- R2, or TNF-R1 (black line) on clearCa-2, -3, -4, and -6 cells was detected by flow cytometry with specific antibodies. Unstained samples are shown in gray. b Expression levels of the DISC proteins c-FLIP, FADD, and caspase-8 as well as caspase-3 in clearCa-2, -3, -4, and -6 cells were analyzed via immunoblotting. Tubulin served as loading control. c Analysis of DNA fragmentation after stimulation of clearCa cells with 0, 2, 4, or 10 ng/mL CD95L in the presence or absence of 10 µg/mL CHX for 16 h. Bars display the mean of at least three experiments, error bars represent SD. Statistical significances were calculated by one-tailed Mann–Whitney U test; * p ≤0.05

Article Snippet: Primary antibodies for specific detection of proteins were: β-Actin (Ac-74, Sigma Aldrich), Bcl-x (Polyclonal, Transduction Laboratories), Caspase-8 (12F5, Dr. Klaus Schulze-Osthoff, Tübingen), Caspase-3 (Polyclonal, R&D Systems), CD95 (C-20, Santa Cruz), c-FLIP (NF6, Adipogen), Cleaved Caspase-3 (Asp175, 9661, Cell Signaling Technology), Cleaved Caspase-8 (18C8, Cell Signaling Technology), FADD (1F7, Upstate), FADD (TA332936, Origene), PARP (4C10–5, BD Biosciences), phospho-Erk1/2 (4370, Cell Signaling Technology), phospho-p65 (3033, Cell Signaling Technology), Tubulin (DM-1A, Sigma Aldrich), XIAP (48, BD Bioscience).

Techniques: Expressing, Cytometry, Western Blot, Control, One-tailed Test, MANN-WHITNEY