cleaved caspase 8  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: DNA-triggered AIM2 condensation orchestrates immune activation and regulation

doi: 10.64898/2026.03.18.712789

Figure Lengend Snippet: (A) AIM2-DNA condensates enrich ASC and caspase-1. Incubation 2 μM DyLight405-labeled ASC and 2 μM AF647-labeled caspase-1(C284A) with condensates formed by 5 μM AIM2 WT or its mutants and 1 μM cy3-labled 100-bp dsDNA in 150 mM NaCl buffer. Scale bar, 10 μm. (B) Cryo-EM images of AIM2-DNA-ASC PYD sample at indicated magnification. Left panel: 2300×, arrowheads indicate AIM2-DNA condensates. Scale bar, 2.5 μm. Right panel: 96000×, arrowheads indicate the ASC PYD filaments. Scale bar, 50 nm. (C) Cryo-ET of AIM2-DNA-ASC PYD sample. Reconstructed tomogram with 3D rendering is shown, gray volume represents AIM2-DNA condensates, green volume represents ASC PYD filaments. Scale bar, 50 nm. (D) Cryo-EM density map of the ASC PYD filament from AIM2-DNA-ASC PYD sample. Left panel: Top view. Middel panel: side view. Right panel: 2D schematic diagram of the ASC PYD assembly. Three types of asymmetric interaction interfaces and helical indexing are shown. (E-F) AIM2-DNA condensates enrich multiple PANoptosome components. (E) Co-incubate 2 μM AF647-labeled ZBP1, 2 μM GFP-RIPK3 RHIM , 2 μM DyLight 405-labeled RIPK1 RHIM-DD with 5 μM AF555-labeled AIM2 and 1 μM 100-bp GC-DNA in 150 mM NaCl buffer. (F) Co-incubate 2 μM DyLight 405-labeled ASC, 2 μM AF488-labeled FADD, 2 μM AF647-labeled caspase-8 DED with 5 μM AF555-labeled AIM2 and 1 μM 100-bp GC-DNA in 150 mM NaCl buffer. Scale bar, 5 μm.

Article Snippet: The membrane was blocked with 5% skim milk in TBST for 1 h and then incubated with indicated primary antibodies overnight at 4°C : anti-AIM2 rabbit pAb (ABclonal, A25252,1:1000), anti-ASC/TMS1 rabbit mAb (CST, 17507, 1:1000), anti-caspase-1 mouse mAb (AdipoGen, AG-20B-0042-C100, 1:1000), anti-GSDMD rabbit mAb (Abcam, ab209845, 1:1000), anti-cleaved caspase-8 rabbit mAb (CST, 8592, 1:1000), anti-caspase-3 rabbit mAb (CST, 14220, 1:1000), anti-cleaved caspase-3 rabbit pAb antibody (CST, 9661, 1:1000), anti-pMLKL rabbit mAb (CST, 37333, 1:1000), anti-pRIPK3 rabbit mAb (CST, 91702, 1:1000), anti-MLKL rabbit mAb (ABclonal, A21894), anti-RIPK3 rabbit mAb (CST, 95702, 1:1000), anti-β-actin mouse mAb (ABclonal, WH278304).

Techniques: Incubation, Labeling, Cryo-EM Sample Prep, Tomography

Journal: bioRxiv

Article Title: DNA-triggered AIM2 condensation orchestrates immune activation and regulation

doi: 10.64898/2026.03.18.712789

Figure Lengend Snippet: (A) Schematic diagram of BMDMs were isolated from mice and cultured to perform assay. (B) Immunofluorescence images of Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. Arrowheads indicate the AIM2-ASC puncta. Scale bar, 10 μm. 4 μm for magnified images. (C) Quantification of the percentage of cells with AIM2-ASC puncta among all cells from (B). These data are representative of three independent experiments. Data are mean ± SEM. Aim2 WT , n=134; Aim2 IDR_M , n=130; Aim2 HIN_M , n=136. Statistical analyses were performed by using two-tailed Student’s t test. *p < 0.05. (D) Immunoblot analysis of pro- and cleaved-caspase-1, pro- and cleaved-GSDMD in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (E) Cell supernatant IL-18 levels in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001. (F) Immunoblot analysis of pro- and cleaved-caspase-1, pro- and cleaved-GSDMD in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 5 h after stimulation with poly(dA:dT). (G) Cell supernatant IL-18 levels in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 5 h after stimulation with poly(dA:dT). These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001. (H) Immunoblot analysis of pro- and cleaved-caspase-8, pro- and cleaved-caspase-3 in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (I) Immunoblot analysis of phosphorylated RIPK3 (p-RIPK3), total RIPK3 (t-RIPK3), phosphorylated MLKL (p-MLKL), total MLKL (t-MLKL) in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (J) Cell death in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. Cell death was measured by SYTOX Green uptake assay. Green indicates dead cells. Scale bar, 50 μm. (K) Quantification of cell death from (J). These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001.

Article Snippet: The membrane was blocked with 5% skim milk in TBST for 1 h and then incubated with indicated primary antibodies overnight at 4°C : anti-AIM2 rabbit pAb (ABclonal, A25252,1:1000), anti-ASC/TMS1 rabbit mAb (CST, 17507, 1:1000), anti-caspase-1 mouse mAb (AdipoGen, AG-20B-0042-C100, 1:1000), anti-GSDMD rabbit mAb (Abcam, ab209845, 1:1000), anti-cleaved caspase-8 rabbit mAb (CST, 8592, 1:1000), anti-caspase-3 rabbit mAb (CST, 14220, 1:1000), anti-cleaved caspase-3 rabbit pAb antibody (CST, 9661, 1:1000), anti-pMLKL rabbit mAb (CST, 37333, 1:1000), anti-pRIPK3 rabbit mAb (CST, 91702, 1:1000), anti-MLKL rabbit mAb (ABclonal, A21894), anti-RIPK3 rabbit mAb (CST, 95702, 1:1000), anti-β-actin mouse mAb (ABclonal, WH278304).

Techniques: Isolation, Cell Culture, Immunofluorescence, Infection, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: DNA-triggered AIM2 condensation orchestrates immune activation and regulation

doi: 10.64898/2026.03.18.712789

Figure Lengend Snippet: (A) Schematic diagram of BMDMs were isolated from mice and cultured to perform assay. (B) Immunofluorescence images of Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. Arrowheads indicate the AIM2-ASC puncta. Scale bar, 10 μm. 4 μm for magnified images. (C) Quantification of the percentage of cells with AIM2-ASC puncta among all cells from (B). These data are representative of three independent experiments. Data are mean ± SEM. Aim2 WT , n=134; Aim2 IDR_M , n=130; Aim2 HIN_M , n=136. Statistical analyses were performed by using two-tailed Student’s t test. *p < 0.05. (D) Immunoblot analysis of pro- and cleaved-caspase-1, pro- and cleaved-GSDMD in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (E) Cell supernatant IL-18 levels in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001. (F) Immunoblot analysis of pro- and cleaved-caspase-1, pro- and cleaved-GSDMD in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 5 h after stimulation with poly(dA:dT). (G) Cell supernatant IL-18 levels in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 5 h after stimulation with poly(dA:dT). These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001. (H) Immunoblot analysis of pro- and cleaved-caspase-8, pro- and cleaved-caspase-3 in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (I) Immunoblot analysis of phosphorylated RIPK3 (p-RIPK3), total RIPK3 (t-RIPK3), phosphorylated MLKL (p-MLKL), total MLKL (t-MLKL) in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. (J) Cell death in Aim2 WT , Aim2 IDR_M and Aim2 HIN_M BMDMs at 16 h after F. novicida infection. Cell death was measured by SYTOX Green uptake assay. Green indicates dead cells. Scale bar, 50 μm. (K) Quantification of cell death from (J). These data from three independent experiments. Data are mean ± SEM. Statistical analyses were performed by using two-tailed Student’s t test. ****p < 0.0001.

Article Snippet: The membrane was blocked with 5% skim milk in TBST for 1 h and then incubated with indicated primary antibodies overnight at 4°C : anti-AIM2 rabbit pAb (ABclonal, A25252,1:1000), anti-ASC/TMS1 rabbit mAb (CST, 17507, 1:1000), anti-caspase-1 mouse mAb (AdipoGen, AG-20B-0042-C100, 1:1000), anti-GSDMD rabbit mAb (Abcam, ab209845, 1:1000), anti-cleaved caspase-8 rabbit mAb (CST, 8592, 1:1000), anti-caspase-3 rabbit mAb (CST, 14220, 1:1000), anti-cleaved caspase-3 rabbit pAb antibody (CST, 9661, 1:1000), anti-pMLKL rabbit mAb (CST, 37333, 1:1000), anti-pRIPK3 rabbit mAb (CST, 91702, 1:1000), anti-MLKL rabbit mAb (ABclonal, A21894), anti-RIPK3 rabbit mAb (CST, 95702, 1:1000), anti-β-actin mouse mAb (ABclonal, WH278304).

Techniques: Isolation, Cell Culture, Immunofluorescence, Infection, Two Tailed Test, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Vinorelbine enhances the efficacy of oncolytic vaccinia virus in a preclinical model of ovarian high-grade serous carcinoma

doi: 10.1016/j.omton.2025.201105

Figure Lengend Snippet: Vaccinia and vinorelbine induces apoptotic ID8 Trp53 −/− cell death (A) Representative phase contrast images of ID8 Trp53 −/− cells after 16 h treatment with DMSO or vinorelbine (1 μM). Scale bars, 150 μm. (B) Immunofluorescent images of microtubule cytoskeleton (tubulin, green) in ID8 Trp53 −/− cells treated with DMSO or vinorelbine with or without infection with ΔVFTK (vaccinia, red). DAPI (blue) was used to stain nuclei and cytoplasmic viral factories. Yellow arrows indicate fragmented nuclei. Scale bars, 10 μM. (C) Immunoblot of the indicated viral proteins following infection for 4 or 8 h with ΔVFTK-NG following 16 h pre-treatment with DMSO or vinorelbine. Vinculin is the cell loading control. (D) Immunoblot examining the levels of LC3 lipidation (LC3-II) and p62 in ID8 Tr53 −/− cells pretreated with vinorelbine or DMSO for 8 h and infected with ΔVFTK-NG for 24 h. Chloroquine (Clq) treatment for 3 h represents the positive control. (E) Immunoblot examining the levels pf MLKL phosphorylation and PARP cleavage in ID8 Tr53 −/− cells pretreated with vinorelbine or DMSO for 8 h and infected with ΔVFTK-NG for 24 h. Staurosporine (S) (8 h) and 0.1% hydrogen peroxide (H 2 O 2 ) combined with Z-VAD (two hours) represent positive controls for apoptosis and necroptosis, respectively. (F) Immunoblot examining the levels of cleaved caspase-8, caspase-3, and PARP in ID8 Tr53 −/− cells treated with vinorelbine 8 h before or after infection with ΔVFTK-NG for 24 h. In (D, E, and F), H5 and GAPDH represent the viral and cell loading controls, respectively. For all experiments, uninfected DMSO-treated cells (UI) were the negative control. Asterisks (∗) indicate non-specific bands. Immunoblot experiments were repeated three times, and representative examples are shown. (G) Median percentage cell confluency over time for the indicated conditions. Cells were treated with the indicated compounds or ΔVFTK-NG at 16 h post-seeding (dashed line numbered 1). The dashed line numbered 2 (24 h after cell seeding) represents the time of addition of either vinorelbine or ΔVFTK-NG for the combination groups.

Article Snippet: Primary antibodies used were F12 (1:4,000, ), F13 (1:6,000, ), H5 (1:10,000, ), GRB2 (1:1,000, Santa Cruz, Dallas, Texas, #sc-255), Vinculin (1:10,000, Sigma-Aldrich, #V9264), GAPDH (1:1,000, Santa Cruz, #sc-32233), PARP (1:1,000, Cell Signaling, Danvers, Massachusetts, #9542), cleaved caspase-8 (1:1,000, Cell Signaling, #8592), cleaved caspase-3 (1:1,000, Cell Signaling, #9664), LC3-B (1:1,000, Abcam [#ab48394], Cambridge, UK), p62/SQSTM1 (1:1,000, Novus Biologicals, Centennial, Colorado, #NBP1-42822), and NeonGreen (1:1,000, Cell Signaling, #41236).

Techniques: Infection, Staining, Western Blot, Control, Positive Control, Phospho-proteomics, Negative Control

Journal: Molecular Therapy Oncology

Article Title: Vinorelbine enhances the efficacy of oncolytic vaccinia virus in a preclinical model of ovarian high-grade serous carcinoma

doi: 10.1016/j.omton.2025.201105

Figure Lengend Snippet: Assessing the efficacy of ΔVFTK-NG-GM-CSF in vivo (A) Schematic representation of the experimental design of the distribution study. Mice were injected i.p. with ID8 Trp53 −/− cells on day 0 and received their i.p. injection on day 28, and the combination groups received their second component (vinorelbine or virus) on day 29. Heat-inactivated (HI) virus (ΔVFTK-NG-GM-CSFi) is the negative control. (B) Quantification of viral DNA in omental tumor, liver, and spleen measured by qPCR and expressed relative to viral DNA levels of the liver sample in the HI virus group. Error bars represent mean ± SD. (C) Representative immunohistochemical images of the distribution of cleaved caspase-3 and NeonGreen in omental tumor, liver, and spleen harvested from a mouse in group 4. The graph shows the quantification of NeonGreen-positive cells in omental tumors. (D) Representative immunohistochemical images of the distribution of cleaved caspase-3 and NeonGreen in omental tumors from mice in groups 1, 3, and 4. The graph shows the quantification of cleaved caspase-3-positive cells in omental tumors. (E) Schematic representation of the experimental design of the vaccinia-vinorelbine combination study. Mice were injected with ID8 Trp53 −/− cells on day 0 and started receiving their i.p. treatment injections on day 21. Mice allocated to single-treatment groups received their inoculations on days 21, 28, and 35, and those allocated to the combination groups received vinorelbine and ΔVFTK-NG-GM-CSF 24 h apart. Heat-inactivated (HI) ΔVFTK-NG-GM-CSFi virus was the negative control. (F) Kaplan-Meier survival curve showing survival data for each group analyzed by log rank test. One mouse belonging to group 2 was excluded from the analysis, as after i.p. injection of ID8 Trp53 −/− cells omental tumor failed to form. The analysis is from the combination of two survival experiments following the same protocols.

Article Snippet: Primary antibodies used were F12 (1:4,000, ), F13 (1:6,000, ), H5 (1:10,000, ), GRB2 (1:1,000, Santa Cruz, Dallas, Texas, #sc-255), Vinculin (1:10,000, Sigma-Aldrich, #V9264), GAPDH (1:1,000, Santa Cruz, #sc-32233), PARP (1:1,000, Cell Signaling, Danvers, Massachusetts, #9542), cleaved caspase-8 (1:1,000, Cell Signaling, #8592), cleaved caspase-3 (1:1,000, Cell Signaling, #9664), LC3-B (1:1,000, Abcam [#ab48394], Cambridge, UK), p62/SQSTM1 (1:1,000, Novus Biologicals, Centennial, Colorado, #NBP1-42822), and NeonGreen (1:1,000, Cell Signaling, #41236).

Techniques: In Vivo, Injection, Virus, Negative Control, Immunohistochemical staining