recombinant human caspase 3 protein  (R&D Systems)

Journal: MedComm

Article Title: Emestrin‐Type Epidithiodiketopiperazines Inhibited Gasdermin D‐Mediated Pyroptosis via Caspase‐3/7 Activation

doi: 10.1002/mco2.70548

Figure Lengend Snippet: Emestrin‐type ETPs inhibited pyroptosis in THP‐1 differentiated macrophages through activation of procaspase‐3/7. (A) Immunoblotting and Coomassie blue staining of cell lysates from THP‐1 differentiated macrophages treated with or without compound 2 (1 µM) 4 h post‐LPS and nigericin induction. P10 band (red rectangle) was sent for mass spectrum analysis. (B) N‑terminal sequence analysis result of p10 band. (C) Mechanism hypothesis of compounds 1 – 11 inhibiting pyroptosis. (D and E) Western blotting analyses of GSDMD, caspase‐3, caspase‐7 cleavage, and α‐tubulin in THP‐1 macrophages treated compound 2 (1 µM) prior and after 4 h pyroptosis induction with LPS and nigericin (mean ± SD; n = 3 per group, t ‐test, ** p < 0.01, **** p < 0.0001). (F) Western blotting analyses of caspase‐1, caspase‐3, caspase‐7 cleavage, and α‐tubulin in the THP‐1 differentiated macrophages treated with each compound (1 µM) 4 h after pyroptosis induction with LPS and nigericin. (G and H) Western blotting analyses of recombinant human procaspase‐3 protein 12 h postincubation with PAC‐1 and each compound (5 µM) respectively at 37 ℃. (I) The IC 50 result of these eleven compounds ( n = 3 per group). (J) In vitro activation of recombinant human procaspase‐3 protein by eleven compounds respectively ( n = 3 per group) and corresponding EC 50 values.

Article Snippet: Recombinant human procaspase‐3 protein (CUSABIO; Cat# CSB‐EP004548HU) was diluted to 200 ng/μL in activation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS).

Techniques: Activation Assay, Western Blot, Staining, Sequencing, Recombinant, In Vitro

Journal: MedComm

Article Title: Emestrin‐Type Epidithiodiketopiperazines Inhibited Gasdermin D‐Mediated Pyroptosis via Caspase‐3/7 Activation

doi: 10.1002/mco2.70548

Figure Lengend Snippet: Emestrin‐type ETPs directly bound to human procaspase‐3 protein. (A) Molecular docking analyses of compounds 1 and 3 – 11 with human procaspase‐3 protein (PDB ID: 3DEK). (B) Molecular docking analysis of compound 2 with human procaspase‐3 protein. (C) Compound binding activity to recombinant human procaspase‐3 protein measured by SPR. (D and E) SPR test of compound 2 interacting with recombinant human procaspase‐3 protein. The K d value is 76.72 µM. (F and G) Western blotting analyses of recombinant human procaspase‐3 protein different time points after incubation with PAC‐1 (5 µM) and compound 2 (5 µM) (mean ± SD; n = 3 per group, t ‐test, ** p < 0.01). (H and I) CETSA results for compound 2 (5 µM) in THP‐1 differentiated macrophage lysates.

Article Snippet: Recombinant human procaspase‐3 protein (CUSABIO; Cat# CSB‐EP004548HU) was diluted to 200 ng/μL in activation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS).

Techniques: Binding Assay, Activity Assay, Recombinant, Western Blot, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: Reprogramming of cancer metabolism via photoresponsive nano-PROTAC enhances pyroptosis-mediated immunotherapy

doi: 10.1038/s41392-025-02405-6

Figure Lengend Snippet: Design of NanoTAC and mechanism of cancer metabolic reprogramming to enhance PDT-mediated pyroptosis. a Chemical structure of the drug conjugate comprising NanoTAC. NanoTAC self-assembles spontaneously from drug conjugates that include an HK2-targeting PROTAC (red), a cathepsin B-cleavable peptide KRR (blue), and the photosensitizer verteporfin (VPF; green) without requiring any additional carrier materials. b Process of NanoTAC-mediated photoimmunotherapy. In a TNBC mouse model, NanoTAC accumulates in tumors and becomes enzymatically activated by lysosomal cathepsin B, inducing pyroptosis via mitochondrial damage upon laser irradiation. This leads to GSDME pore formation, as activated caspase-3 (CASP3) cleaves GSDME-FL into GSDME-NT fragments, resulting in the extracellular release of DAMPs and inflammatory cytokines. Moreover, PROTAC-mediated TPD eliminates intracellular HK2, which independently damages mitochondria and counteracts glucose and oxygen consumption during the photodynamic process. c This synergistic combination of PDT and PROTAC triggers significantly amplified pyroptotic tumor cell death, drives the cancer-immunity cycle to recruit effector cells, and ultimately inhibits primary, metastatic, and recurrent TNBC

Article Snippet: To assess target biomarker-specific cleavage, NanoTAC at pH 5.5 2-(N-morpholino)ethanesulfonic acid (MES) buffer was incubated at 37 °C with Cat-B (1, 2, 5 or 10 μg; Cat. No. 953CY, R&D systems), Cat-B with Z-FA-FMK (Cat. No. HY-P0109A, MedChemExpress), Cat-D (Cat. No. 1014-AS, R&D Systems), Cat-E (Cat. No. 1294-AS, R&D Systems), Cat-L (Cat. No. 952-CY, R&D Systems), MMP-9 (Cat. No. 911-MP, R&D Systems) or CASP3 (Cat. No. 707-C3/CF, R&D Systems), followed by LC/MS analysis.

Techniques: Irradiation, Amplification

Journal: Signal Transduction and Targeted Therapy

Article Title: Reprogramming of cancer metabolism via photoresponsive nano-PROTAC enhances pyroptosis-mediated immunotherapy

doi: 10.1038/s41392-025-02405-6

Figure Lengend Snippet: Physicochemical Characterization of NanoTAC. a Electrostatic potential surface and schematic representation of the drug conjugate comprising NanoTAC. b Representative MD simulation snapshots showing the time-dependent self-assembly behavior of the drug conjugates in an explicit water box. c Classification of major noncovalent interactions observed at 50 ns of simulation time. d Molecular surface model of NanoTAC and solvent accessibility mapping of individual molecular domains, presented by component and total surface areas. e Size distribution profiles of NanoTAC. f TEM image of NanoTAC. Scale bar, 500 nm. g Particle stability of NanoTAC in mouse serum. Changes in particle size and polydispersity index over time were analyzed using dynamic light scattering (DLS; n = 5). h UV‒vis absorbance and i fluorescence (FL) spectra of the indicated compounds. j ROS generation by NanoTAC and VPF under laser irradiation at a power of 40 mW (n = 5). Cleavage behavior of NanoTAC after incubation in k Cat-B alone or in combination with Z-FA-FMK or l other enzymes, including Cat-E, Cat-L, Cat-D, CASP3, and MMP-9. m Changes in the binding affinity of NanoTAC for HK2 or VHL before and after cleavage. Statistical significance was determined by Student’s t-test ( j ) and one-way ANOVA with Tukey‒Kramer post hoc test ( g )

Article Snippet: To assess target biomarker-specific cleavage, NanoTAC at pH 5.5 2-(N-morpholino)ethanesulfonic acid (MES) buffer was incubated at 37 °C with Cat-B (1, 2, 5 or 10 μg; Cat. No. 953CY, R&D systems), Cat-B with Z-FA-FMK (Cat. No. HY-P0109A, MedChemExpress), Cat-D (Cat. No. 1014-AS, R&D Systems), Cat-E (Cat. No. 1294-AS, R&D Systems), Cat-L (Cat. No. 952-CY, R&D Systems), MMP-9 (Cat. No. 911-MP, R&D Systems) or CASP3 (Cat. No. 707-C3/CF, R&D Systems), followed by LC/MS analysis.

Techniques: Solvent, Fluorescence, Irradiation, Incubation, Binding Assay