SMAC (AT-406) (#HY-15454) and z-VAD(OMe)-FMK (#HY-16658) were purchased from MedChemExpress LLC (Shanghai, China). Sytox green (SYTOX® Green Nucleic Acid Stain) (#S7020) was purchased from Invitrogen Inc. (Carlsbad, CA, USA). FITC Annexin V Apoptosis Detection Kit (Cat# 556547) was purchased from BD Pharmingen (Shanghai, China). Antibodies against human phospho-RIPK1S166 (#65746), murine phospho-RIPK1S166 (#31122), RIPK1 (#3493), human phospho-RIPK3S227 (#93654), murine RIPK3 (#95702), human RIPK3 (#13526), murine MLKL (#37705), human MLKL (#14993), caspase-3 (#14220), cleaved caspase-3 (#9664), caspase-8 (#4790), cleaved human caspase-8 (#9496S), cleaved mouse caspase-8 (#8592), PARP (#9532S), and anti-FLAG antibody (#8146) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against β-actin (#sc-47778) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology Inc. (San Diego, CA, USA). Antibodies against murine phospho-MLKL (#ab196436), human phospho-MLKLS358 (#ab187091), and murine phospho-RIPK3T231/S232 (#ab222320) were purchased from Abcam Inc. (Shanghai, China). The anti-GFP (Code#HT801 Lot#O21209) mouse monoclonal antibody was purchased from TransGen Biotech (Beijing, China). Recombinant murine TNF-α (#410-MT-010) was purchased from R and D Systems, Inc. (Shanghai, China).
Z vad ome fmk
Manufactured by MedChemExpress
Sourced in United States
Z-VAD(OMe)-FMK is a broad-spectrum caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes. It is a widely used tool in the study of apoptosis and programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Antibodies against β actin, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal antibody, by Transgene (1 mentions)
Anti flag antibody, by Cell Signaling Technology (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Mtt reagent, by Thermo Fisher Scientific (1 mentions)
C4859, by Merck Group (1 mentions)
Mtt colorimetric assay, by Thermo Fisher Scientific (1 mentions)
Streptozocin stz, by Merck Group (1 mentions)
Int 777, by MedChemExpress (1 mentions)
Mdivi 1, by MedChemExpress (1 mentions)
3 methyladenine, by MedChemExpress (1 mentions)
Mitobright lt red, by Dojindo Laboratories (1 mentions)
Tomm20, by Proteintech (1 mentions)
Phospho drp1, by Cell Signaling Technology (1 mentions)
Pink1, by Cell Signaling Technology (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Evans blue, by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
5 protocols using z vad ome fmk
1
Apoptosis and Necroptosis Pathway Analysis
2
Inhibition of Cell Death Signaling
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Cells were incubated with different combination of a pan-caspase inhibitor, Z-VAD(OMe)-FMK (20 μmol/L, #HY-16658, MedChemExpress, NJ, USA), a cIAP1/2 inhibitor, BV-6 (10 μmol/L, #HY-16701, MedChemExpress) and an inhibitor of protein synthesis, cycloheximide (CHX) (0.5 μg/mL, #C4859, Sigma-Aldrich) 1 h before TNF-α stimulation (20 ng/mL, #315-01A, PeproTech, Neuilly-sur-Seine, Paris). Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTT) colorimetric assay (Thermo Fisher Scientific), which measures cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH) to reduce the MTT to its insoluble formazan end product, which has a purple color. Cells were incubated with 0.5 mg/mL of MTT reagent (Thermo Fisher Scientific) for 2 h. Once MTT crystals were developed and controlled under light microscopy, they were dissolved in DMSO and quantified by measuring absorbance at 540 nm.
3
Mitochondrial Dynamics Modulation in Diabetic Cardiomyopathy
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Mdivi-1, 3-bromopyruvic acid, INT-777, 3-methyladenine, Rapamycin, BAPTA-AM, and Z-VAD (OMe)-FMK were procured from MedChemExpress (NJ, United States). MitoBright LT Red was obtained from DOJINDO (Kumamoto, Japan). Glucose, streptozocin (STZ) and Evans Blue were obtained from Sigma-Aldrich (St. Louis, United States). Fluo-4 AM, Cell counting kit-8 (CCK8), JC1 assay kit and cell mitochondria isolation kit were obtained from Beyotime (Shanghai, China). The TUNEL assay and haematoxylin-eosin (HE) staining kits were obtained from Roche (Mannheim, Germany) and BOSTER (Wuhan, China), respectively. The primary antibodies used were as follows: PKC δ, Phospho-PKCδ, HK2, LC3B, P62, DRP1 (Abcam, Cambridge, United States); Parkin, PINK1, Phospho-DRP1, Cleaved caspase-3 (Cell Signaling Technology, Danvers United States), Bax, β-actin, COX4 (Proteintech, Wuhan, China); and TOMM20 (Santa, CA, United States). information on reagents and antibodies is shown in Supplementary Tables S1, S2 .
4
Cytotoxicity Evaluation of Compounds
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Cells were plated in 96-well plate at the density of 1 × 104 per well and were allowed to attach for 24 h 5 μM Sorafenib (Cell Signaling Technology, USA), 5 μM ferrostatin-1 (S7243, Selleck, USA), 50 μM deferoxamine (S5742, Selleck, USA), 10 μM Z-VAD(OMe)-FMK (HY-16658, MedChemExpress, USA) or 1 μM Necrosulfonamide (HY-100573, MedChemExpress, USA)were added to the cells as indicated in Figure legends and incubated for 24 h. The cells were harvested and Cytotoxicity LDH assays were performed using the Cytotoxicity LDH assay kit (CK12, Dojindo, Japan) according the manufacturer's instructions as previously described [45 (link)].
5
Small Molecule Inhibitors for Cellular Studies
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The following small compound inhibitors and reagents were used: 2-Deoxy-D-glucose (Sigma-Aldrich), anisomycin (Sigma-Aldrich), bafilomycin A1 (Sigma-Aldrich), berzosertib (Selleckchem), bestatin methyl ester (Abcam), blasticidin (InvivoGen), bortezomib (Selleckchem), cycloheximide (Sigma-Aldrich), deoxyribonucleic acid sodium salt from herring testes (Sigma-Aldrich), doramapimod (Cayman), doxycycline (Thermo Fisher Scientific), doxorubicin (Selleckchem), etoposide (Calbiochem), H2O2 (Roth), ISRIB (Selleckchem), JNK-IN-8 (Selleckchem), KU-60019 (Selleckchem), lactimidomycin (Sigma-Aldrich), LPS-EK Ultrapure (Invivogen), MG-132 (Selleckchem), MLN4924 (MedChem Express), MLN7243 (ChemieTek), Nigericin sodium salt (Biomol), PF-3644022 (Sigma-Aldrich), SB202190 (Sigma-Aldrich), poly(dA:dT) (Invivogen), poly(I:C) low molecular weight and high molecular weight (Invivogen), sodium azide (Sigma-Aldrich), talabostat mesylate (MedChemExpress), Vx-765 (Selleckchem), and Z-VAD(Ome)-FMK (MedChemExpress). ZAKα inhibitor 6p (Yang et al., 2020 (link)) was kindly provided by IFM Therapeutics. 6p was synthesized and characterized by 1H NMR spectroscopy and LC-MS, with the data being fully consistent with literature values for this compound.
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