anti cleaved caspase 9  (Cell Signaling Technology Inc)

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs preferentially induced mtApoptosis and immunogenic cell death in cancer cells (A) Flow cytometry analysis of cells positive for Annexin V and propidium iodide (PI) in cancer and non-cancer cell lines after treatment with PBS (control), NPs, naked Puma mRNA, and mPuma@NPs for 12 h ( n = 3). (B) Western blot analysis of mitochondrial apoptois pathway in CT-26 cells treated with control, NPs, mPuma@NPs, and mBim@NPs. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, cleaved caspase-3 (C-Cas3), caspase9, and cleaved caspase-9 (C-Cas9) proteins were detected. β-Actin was used as the loading control ( n = 3). (C) Confocal laser scanning microscopy (CLSM) images of the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) probe in CT-26 cells after treatment with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h. Increased JC-1 monomer signal (green) and decreased JC-1 aggregate signal (red) indicate a decrease in mitochondrial membrane potential ( n = 3). Scale bars, 200 μm. (D) Flow cytometry analysis of cellular oxygen species (ROS) levels using 2,7-dichlorofluorescein diacetate (DCFH-DA) staining in CT-26 cells after 12 h incubation with PBS (control), NPs, naked mRNA, mPuma@NPs, and mBim@NPs ( n = 3). (E) CLSM images of CRT expression in B16-F10 and CT-26 cells after 12 h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). Scale bars, 20 μm. (F) Extracellular ATP and HMGB1 expression levels were analyzed by ELISA in B16-F10 cells after 12-h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). (G) Flow cytometry analysis and quantification of immune stimulation in BMDCs co-cultured with B16-F10 cells pretreated with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h, followed by 48-h co-culture ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Cleaved-Caspase 9 (Asp353) Antibody , Abmart , Cat#TA5240S; RRID:AB_3740854.

Techniques: Flow Cytometry, Control, Western Blot, Confocal Laser Scanning Microscopy, Membrane, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

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

Journal: Pancreas

Article Title: Knockdown of Lymphoid Enhancer-binding Factor 1 Inhibits Pancreatic Adenocarcinoma Growth and Neoangiogenesis by Curbing Notch1 and Nuclear Factor Kappa B Signaling Pathways

doi: 10.1097/MPA.0000000000002562

Figure Lengend Snippet: Knockdown of LEF1 induces apoptosis in PAAD cells. A–C, Flow cytometry detection of apoptosis and apoptosis rate statistics: apoptosis rate was higher in AsPC-1/shLEF1 and BxPC-3/shLEF1. D–F, Calcein AM/PI staining assay results and statistics: the live cells decreased while the dead cells increased in AsPC-1/shLEF1 and BxPC-3/shLEF1. G, ATP production assay: the ATP production decreased in AsPC-1/shLEF1 and BxPC-3/shLEF1. H, LDH release assay showed an increase in LDH release in AsPC-1/shLEF1 and BxPC-3/shLEF1. I–M, WB results and quantitative analysis: the level of Bcl-2 protein decreased in AsPC-1/shLEF1 and BxPC-3/shLEF1, while BAX, Cleaved caspase-3 and Cleaved caspase-9 increased (n = 3, * P <0.05, ** P <0.01).

Article Snippet: The first antibodies were as follows: LEF1 (ab137872, 1:1000, Abcam), proliferating cell nuclear antigen (PCNA, ab29, 1:1000, Abcam), MMP-2 (4022S, 1:1000, CST), MMP-9 (3852S, 1:1000, CST), BAX (2772S, 1:1000, CST), Bcl-2 (15071S, 1:1000, CST), Cleaved caspase-3 (9661S, 1:1000, CST), Cleaved caspase-9 (9509S, 1:1000, CST), vascular endothelial growth factor-A (VEGFA, ab214424, 1:1000, Abcam), total Notch1 (ab52627, 1:1000, Abcam), NICD (4147, 1:1000, CST), Hes1 (ab71559, 1:1000, Abcam), Hey1 (ab235173, 1:1000, Abcam), Jagged1 (ab300561, 1:1000, Abcam), P65 (ab32536, 1:1000, Abcam), p-P65 (3033S, 1:1000, CST), GAPDH (5174S, 1:5000, CST), HRP-anti-Rabbit (A0362, 1:1000, Beyotime), HRP-anti-Mouse (A0350, 1:1000, Beyotime).

Techniques: Knockdown, Flow Cytometry, Staining, Lactate Dehydrogenase Assay