cleaved casp3  (Cell Signaling Technology Inc)

Journal: Cancer Cell International

Article Title: NSC632839 suppresses esophageal squamous cell carcinoma cell proliferation in vitro by triggering spindle assembly checkpoint-mediated mitotic arrest and CREB-Noxa-dependent apoptosis

doi: 10.1186/s12935-025-03831-w

Figure Lengend Snippet: NSC632839 treatment induces intrinsic apoptosis. ( A ) AnnexinV-FITC/PI double stained kit was used to detect NSC632839-induced apoptosis. Cells were incubated with NSC632839 for 48 h, collected and stained with AnnexinV-FITC and PI for apoptosis detection using FACS. The cells in the upper and lower right quadrant represent Annexin V + cells, which were defined as apoptotic cells. Representitive images were shown in left panel. Statistical analysis was shown in the right panel. ( B ) Detecting the effect of NSC632839 on caspase3 activity. Cells were incubated with NSC632839 for 48 h. Then cells were collected and caspase3 activity was evaluated by CaspGLOW Fluorescein Active Caspase3 Staining kit. Representitive images were shown in left panel. The cell numbers with positive caspase3 activity were calculated, statistical analysis and shown in the right panel. ( C ) Evaluation of mitochondrial membrane depolarization. Mitochondrial membrane depolarization was analyzed by flow cytometry and displayed as in left panel. Cells that had lost mitochondrial membrane potential (MMP) appeared in the right lower quadrant, which were calculated, analyzed and shown in right panel. ( D ) Detection of apoptosis-related proteins using immunoblot assay

Article Snippet: For simultaneous detection of CASP3 activity and p-H3, before cell fixation, the cells were incubated with GreenNuc Caspase-3 Substrate at room temperature to avoid light for 1 h (Beyotime, China).

Techniques: Staining, Incubation, Activity Assay, Membrane, Flow Cytometry, Western Blot

Journal: Cancer Cell International

Article Title: NSC632839 suppresses esophageal squamous cell carcinoma cell proliferation in vitro by triggering spindle assembly checkpoint-mediated mitotic arrest and CREB-Noxa-dependent apoptosis

doi: 10.1186/s12935-025-03831-w

Figure Lengend Snippet: NSC632839-induced M phase arrest happens prior to apoptosis. ( A - E ) Cells were incubated with NSC632839 and collected at indicated time for detecting apoptosis ( A - B ) or cell cycle ( C - D ) by FACS. Cell proteins were collected to evaluate the expression of p-H3 (Ser 10) and cleaved-PARP ( E ). ( F ) Apoptotic cells come from M-phase-arrested cell in NSC632839-treated cells. After treated with NSC632839 or DMSO, the expression of p-H3 and the activation of caspase3 was evaluated by immunofluorescence as described in Material and Methods. Scale bar = 100 μm

Article Snippet: For simultaneous detection of CASP3 activity and p-H3, before cell fixation, the cells were incubated with GreenNuc Caspase-3 Substrate at room temperature to avoid light for 1 h (Beyotime, China).

Techniques: Incubation, Expressing, Activation Assay, Immunofluorescence

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A Progress curves reporting on the purified Cg CASP3 processing of the caspase-specific substrates (Ac-YVAD-pNA (caspase-1), Ac-VDQQD-pNA (caspase-2), Ac-DEVD-pNA (caspase-3/7), Ac-LEVD-pNA (caspase-4), Ac-VEID-pNA (caspase-6), Ac-IETD-pNA (caspase-8) and Ac-LEHD-pNA (caspase-9)) as a function of time in caspase activity buffer with 50 μM PAC-1 ( n = 5). B Progress curves reporting on the purified Cg CASP3 processing of the caspase-3/-7 substrate (Ac-DEVD-pNA) as a function of time in the presence of universal caspases inhibitor (Z-VAD-FMK) or caspase-3/-7 inhibitor Ac-DEVD-CHO ( n = 5). C Coomassie staining of purified Cg CASP3 and its long IL deletion mutant ( Cg CASP3-∆M) treated with 50 μM PAC-1 for 120 min. The cell-based transfection DEVD-ase activity assay ( D .; n = 5), the CCK-8 (Cell Counting Kit-8) assay for the cell viability ( E .; n = 5), the lactate dehydrogenase (LDH) release assay ( F .; n = 5) and the cell apoptosis rate ( G .; n = 3; the left panel is the cell apoptosis chart in each group, and the right panel is the cell apoptosis rate in each group) of HEK293T cells were transfected with mCherry- CgCasp3 or mCherry- CgCasp3 -∆M, and incubated with TNF-α + SM-164 for 8 h. The control group is the HEK293T cells transfected with the pCMV-N-mCherry empty plasmid. H The western blotting of HEK293T cells transfected with mCherry- CgCasp3 or mCherry- CgCasp3 -∆M, and incubated with TNF-α + SM-164 for 8 h. I Progress curves reporting on the purified Cg CASP3 and Cg CASP3-∆M processing of the caspase-3/-7 substrate (Ac-DEVD-pNA) as a function of time in caspase activity buffer with 50 μM PAC-1 ( n = 5). The error bars represent the S.D. Significant differences among groups were marked with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. “ns” indicates non-significant differences.

Article Snippet: After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Techniques: Purification, Activity Assay, Staining, Mutagenesis, Transfection, CCK-8 Assay, Cell Counting, Lactate Dehydrogenase Assay, Incubation, Control, Plasmid Preparation, Western Blot

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A The phosphorylation levels of Thr260 site of Cg CASP3 in gill tissues of C. gigas and C. angulata during heat stress, which was obtained from our previous study . B The DEVD-ase activity assay of C. gigas and C. angulata during heat stress ( n = 3). C The sequence alignment surrounding T260 on Cg CASP3. The cell-based transfection DEVD-ase activity assay ( D .; n = 5), the CCK-8 assay for the cell viability ( E .; n = 5), the lactate dehydrogenase (LDH) release assay ( F .; n = 5) and the cell apoptosis rate ( G .; n = 3; The left panel is the cell apoptosis chart in each group, and the right panel is the cell apoptosis rate in each group) of HEK293T cells were transfected with mCherry- CgCasp3 , mCherry- CgCasp3 T260A (mimicking dephosphorylation) or mCherry- CgCasp3 T260D (mimicking phosphorylation), and incubated with TNF-α + SM-164 for 8 h. The control group is the HEK293T cells transfected with the pCMV-N-mCherry empty plasmid. There were no significant differences among the four groups in panels ( D ) (0, 2, 4 h) and E (8 h). H The western blotting of HEK293T cells transfected with mCherry- CgCasp3 , mCherry- CgCasp3 T260A or mCherry- CgCasp3 T260D , and incubated with TNF-α + SM-164 for 8 h. I Progress curves reporting on the purified Cg CASP3, Cg CASP3 T260A and Cg CASP3 T260D processing of the caspase-3/-7 substrate (Ac-DEVD-pNA) as a function of time in caspase activity buffer with 50 μM PAC-1 ( n = 5). The error bars represent the S.D. Significant differences among groups were marked with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. “ns” indicates non-significant differences.

Article Snippet: After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Sequencing, Transfection, CCK-8 Assay, Lactate Dehydrogenase Assay, De-Phosphorylation Assay, Incubation, Control, Plasmid Preparation, Western Blot, Purification

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A Co-immunoprecipitation (co-IP) of Cg CASP3 with Cg AKT. HEK293T cells expressing the indicated constructs encoding mCherry- CgCasp3 and Myc- CgAkt were lysed and incubated with anti-mCherry magnetic beads overnight, Myc-tagged molecules co-IPed in this manner were resolved by SDS-PAGE and detected by immunoblotting with anti-Myc antibody. The expression of Cg CASP3 and Cg AKT by transfectants (INPUT) in these studies was also confirmed by immunoblot analysis. B Yeast two-hybrid assay between Cg CASP3 and Cg AKT. Full-length of CgCasp3 and CgAkt were fused to the pGBKT7 binding domain (BD, bait) and the pGADT7 activation domain (AD, prey), respectively, and then transformed into yeast. Shown are growth phenotypes of yeast transformants on selective media of SD/Leu-Trp- (left panel), SD/Leu-Trp-His- (central panel, interaction) and SD/Leu-Trp-His-Ade2 (right panel, interaction). C BiFC assay of Cg CASP3 with Cg AKT. HeLa cells were transfected BiFC plasmids expressing Cg CASP3 (pBiFC-VN173- CgCasp3 ) only, Cg AKT (pBiFC-VC155- CgAkt ) only or Cg CASP3 and Cg AKT. Images were acquired with a confocal microscope at the EGFP channel. Bar: 10 µm. D Subcellular localization of Cg CASP3 and Cg AKT in HeLa cells under control and heat stress. HeLa cells were transfected mCherry- CgCasp3 and Myc- CgAkt . Images were acquired with a confocal microscope under control and heat treatment. Bar: 10 µm. E In vitro kinase activity assay of Cg AKT on Cg CASP3 Thr260 site. The recombinant proteins (His- Cg AKT and His- Cg CASP3/His- Cg CASP3 T260A ) were incubated in kinase buffer at 30 °C for 30 min and detected by western blotting with anti-His and anti-phosphoserine/threonine (anti-pS/pT) antibodies. F In vivo phosphorylation assay of Cg AKT on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 /mCherry- CgCasp3 T260A and Myc-Cg Akt . The cells were lysed and incubated with anti-mCherry magnetic beads overnight, and IPed proteins were separated using SDS-PAGE gels. The phosphorylation level of Cg CASP3/ Cg CASP3 T260A were detected by western blotting using anti-phosphoserine/threonine antibody. The cell-based transfection DEVD-ase activity assay ( G .; n = 5), the CCK-8 assay for the cell viability ( H .; n = 5), the lactate dehydrogenase (LDH) release assay ( I .; n = 5) and the cell apoptosis rate ( J .; n = 3) of HEK293T cells were transfected with mCherry- CgCasp3 /mCherry- CgCasp3 T260A and Myc- CgAkt , and incubated with TNF-α + SM-164 for 8 h ( n = 3). The error bars represent the S.D. Significant differences among groups were marked with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Construct, Incubation, Magnetic Beads, SDS Page, Western Blot, Y2H Assay, Binding Assay, Activation Assay, Transformation Assay, Bimolecular Fluorescence Complementation Assay, Transfection, Microscopy, Control, In Vitro, Kinase Assay, Recombinant, In Vivo, Phospho-proteomics, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A The sequence alignment surrounding T291 on oyster AKT protein. The result showed that the oyster AKT residues T291 correspond to the conserved active sites T308/T309/T305 in human AKT1/AKT2/AKT3. B The western blotting of proteins extracted from gill tissues of C. gigas and C. angulata during heat stress with PI3K-AKT pathway’s antibodies. Gi and An represent the C. gigas and C. angulata under control condition. HGi and HAn represent the C. gigas and C. angulata under heat stress. C In vivo phosphorylation assay of different Cg AKT mutants on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A /Myc- CgAkt T291DS464D . The cells were lysed and incubated with anti-mCherry magnetic beads overnight, and IPed proteins were separated using SDS-PAGE gels. The phosphorylation level of Cg CASP3 were detected by western blotting using anti-phosphoserine/threonine antibody. D In vitro kinase activity assay of Cg AKT/ Cg AKT T291AS464A on Cg CASP3 Thr260 site. The recombinant proteins (His- Cg AKT/His- Cg AKT T291AS464A /His- Cg AKT T291DS464D and His- Cg CASP3) were incubated in kinase buffer at 30 °C for 30 min and detected by western blotting with anti-His and anti-phosphoserine/threonine antibodies. The western blotting ( E ), The cell-based transfection DEVD-ase activity assay ( F .; n = 5), the CCK-8 assay for the cell viability ( G .; n = 5), the lactate dehydrogenase (LDH) release assay ( H .; n = 5) and the cell apoptosis rate ( I .; n = 3) of HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A /Myc- CgAkt T291DS464D , and incubated with TNF-α + SM-164 for 8 h. J In vivo phosphorylation assay of Cg AKT and its upstream regulators in PI3K-AKT pathway ( Cg PDPK1, Cg PIK3CA and Cg PTEN) on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A , His- CgPdpk1 , HA- CgPik3ca and Flag- CgPten . The cells were lysed and incubated with anti-mCherry magnetic beads overnight, and IPed proteins were separated using SDS-PAGE gels. The phosphorylation level of Cg CASP3 were detected by western blotting using anti-phosphoserine/threonine antibody. K The western blotting of HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A , His- CgPdpk1 , HA- CgPik3ca and Flag- CgPten , and incubated with TNF-α + SM-164 for 8 h. The error bars represent the S.D. Significant differences among groups were marked with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Techniques: Sequencing, Western Blot, Control, In Vivo, Phospho-proteomics, Transfection, Incubation, Magnetic Beads, SDS Page, In Vitro, Kinase Assay, Recombinant, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: Under thermal stress, the PI3K/AKT pathway is activated via RTKs (Receptor Tyrosine Kinases) or ITGs (Integrins). Then, upregulated Cg PI3K complex in C. angulata catalyzes PIP 2 to PIP 3 , which recruits Cg PDK1 (the phosphorylation level of the Ser184 autophosphorylation site is increased in C. angulata ) to phosphorylates Cg AKT Thr291 site. Additionally, the phosphatase PP2A/PTEN, dephosphorylating the Thr291 sites of Cg AKT/dephosphorylating phosphoinositides to suppress the PI3K-AKT pathway, also shows stronger downregulation in C. angulata , indicating its involvement in regulating the higher kinase activity of Cg AKT in C. angulata . Finally, stronger activated Cg AKT phosphorylates Cg CASP3 Thr260 site to inhibit its heat-induced activation to shape lower CASP3 enzyme activity and stronger thermal resistance of C. angulata .

Article Snippet: After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Activation Assay