Thiazolidinediones (rosiglitazone, pioglitazone, ciglitazone) and the PPARγ antagonist GW9662 were provided from Cayman Chemical (Ann Arbor, MI, USA). Soluble recombinant TRAIL, proteasome inhibitor MG132, caspase 9 specific inhibitor (Z-LEHD-FMK) and propidium iodide (PI) were purchased from Sigma (Saint Quentin Fallavier, France). Caspase 8 inhibitor (Z-IETD-FMK) was from Bachem (Heidelberg, Germany). Anti-DR4 and DR5 blocking antibodies were from Alexis Biochemicals (Lausanne, Switzerland).
Z lehd fmk
Manufactured by Merck Group
Sourced in United States
Z-LEHD-FMK is a synthetic peptide compound that functions as a caspase-3 inhibitor. It is commonly used in research applications to investigate apoptosis and cell death pathways.
Automatically generated - may contain errors
Lab products found in correlation
Z devd fmk, by Merck Group (11 mentions)
Z ietd fmk, by Merck Group (10 mentions)
Z vad fmk, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
3 methyladenine 3 ma, by Merck Group (2 mentions)
Rabbit anti human β actin antibody, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Rhodamine 123, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Pioglitazone, by Cayman Chemical (1 mentions)
Ciglitazone, by Cayman Chemical (1 mentions)
Gw9662, by Cayman Chemical (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
Oleandrin, by Merck Group (1 mentions)
23 protocols using z lehd fmk
1
Apoptosis Induction by PPARγ Antagonists
2
Oleandrin Cytotoxicity Mechanism Investigation
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Oleandrin was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The molecular structure of Oleandrin is showed in the Supplementary file (Figure S1) . The purity was approximately 99%, as analyzed by HPLC. A 1 mmol/L stock solution (Mr = 576.73) was prepared by dissolving Oleandrin in 100% DMSO (Sigma-Aldrich) and was stored at −80 °C. All subsequent dilutions were made in the medium. The final concentration of DMSO was confirmed to be less than 0.1%. The pan-caspase inhibitor with cell membrane permeability, z-VAD-fmk, was purchased from Beyotime (Beyotime Biotech, Nanjing, China). The caspase-9 inhibitor, z-LEHD-fmk, was obtained from Sigma (Sigma-Aldrich Chemical Co.). The Fas blocking antibody (Abcam, Cambridge, MA, USA) was kindly donated by Wei Zhang from the Institute of Zoology, Chinese Academy of Science.
3
Optimizing Cell Survival Pathways
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4
Autophagy and Caspase Inhibition in Cells
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For the autophagy inhibition, 3-methyladenine (3-MA) (Sigma, St Louis, MO M921) was used at 5mM and 10 mM concentrations and chloroquine diphosphate (Sigma, St Louis, MO) was used at 50 μ;M and 100 μ;M. Cells were exposed to these inhibitors for 20 minutes prior to treatment with either DMSO or VMY.
The caspase 8 inhibitor Z-IETD-FMK, (Sigma, St Louis, MO) and the caspase 9 inhibitor Z-LEHD-FMK (Sigma, St Louis, MO) were resuspended in DMSO and used at final concentrations of 20uM.
The caspase 8 inhibitor Z-IETD-FMK, (Sigma, St Louis, MO) and the caspase 9 inhibitor Z-LEHD-FMK (Sigma, St Louis, MO) were resuspended in DMSO and used at final concentrations of 20uM.
5
Apoptosis-related Protein Inhibition Assay
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For blockage test, the cell monolayers were pretreated with 10 μM cyclosporine A (CSA, a blocker of mitochondrial permeability transition (MPT), Sigma) for 30 min, 50 μM Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhitor, Sigma) for 1 h at 37°C (Jin et al., 2009 (link); Hu et al., 2013 (link)), 25 μM LY294002 (PI3K/Akt pathway inhibitor, Sigma) for 1 h at 37°C, or 10 mM N-acetyl-cysteine (NAC, an anti-oxidant, Sigma) for 1 h at 37°C. In these tests, macrophages without blocker pretreatment were used as the controls. For RNA interference test, small interfering RNAs (siRNAs) specific to silence the mouse or human bid, aif, and endoG genes were designed and synthesized by Invitrogen Co. (Shanghai, China). The sequences of siRNAs are 5′-CGACUGUCAACUUUAUUAATT-3′ in J774A.1 and 5′-GGCCACUGUUUGGAAAUAATT-3′ in THP-1 (targeting bid); 5′-GGAAAUAUGGGAAAGAUCCTT-3′ in J774A.1 and 5′-GGCUACGUCCAGGAGCGCACC-3′ in THP-1 (targeting aif); 5′-GGACCGAGGCTGATGGGAA-3′ in J774A.1 and 5′-AAGAGCCGCGAGUCGUACGUG-3′ in THP-1 (targeting endoG). The transfection was performed using Lipofectamine-2000 (Invitrogen), according to the manufacturer's protocol.
6
Acetylshikonin Induces Apoptosis in HCC Cells
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Human hepatocellular carcinoma cells HepG2, Huh7 and normal hepatic cell LO2 were provided by Dr. Wu in The University of Kansas. Wild-type and p53−/−Hep3B cells, wild-type and p53 upregulated modulator of apoptosis (PUMA)−/−Hep3B cells, HepG2 Bax+/− and HepG2 Bax−/− Cells were provided by Prof. Shi in the University of Kansas. Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with penicillin (5 units/mL), streptomycin (5 ug/mL), and 10% heat-inactivated fetal bovine serum (FBS). Acetylshikonin was obtained from Huakang Pharmaceutical Company (Deyang, China) and resolved in 0.1% dimethyl sulfoxide (DMSO). zlETD-fmk, zlEHD-fmk, zDEVD-fmk and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich Corp (St. Louis, MO, USA). Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was purchased from R&D Systems (Wiesbaden, Germany) and resolved in PBS + 0.01% bovine serum albumin (BSA).
7
Isolating and Infecting Headkidney Macrophages
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The fish were euthanized using MS 222 (Sigma), headkidney excised aseptically and HKM were isolated using 34/51% percoll gradient as described earlier (33 (link)). The HKM were infected with A. hydrophila (MOI 1:50) for 1 h and extracellular bacteria was removed using chloramphenicol (30 µg/mL) as described earlier (16 (link)).
The HKM were pre-incubated separately with mPTP inhibitor [Cyclosporin A (CsA), 5 µM, Sigma], mitochondrial Ca2+ uniporter (MCU) inhibitor [Ruthenium Red (RR), 20 µM, Sigma], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Sigma], DRP-1 inhibitor [Mdivi-1, 25 µM, Sigma], HIF-1α inhibitor [Dimethyl-bisphenol A (di-BPA), 200 µM, Abcam], mtROS inhibitor [YCG063, 10 µM, Calbiochem], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Biovision], caspase-3 inhibitor [Ac-DEVD-CHO, 10 µM, Sigma], for 1 h and then infected with A. hydrophila as mentioned earlier (16 (link)). The inhibitor concentrations used in the study had no adverse effects on the viability of HKM and bacterial growth per se (data not shown).
The HKM were pre-incubated separately with mPTP inhibitor [Cyclosporin A (CsA), 5 µM, Sigma], mitochondrial Ca2+ uniporter (MCU) inhibitor [Ruthenium Red (RR), 20 µM, Sigma], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Sigma], DRP-1 inhibitor [Mdivi-1, 25 µM, Sigma], HIF-1α inhibitor [Dimethyl-bisphenol A (di-BPA), 200 µM, Abcam], mtROS inhibitor [YCG063, 10 µM, Calbiochem], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Biovision], caspase-3 inhibitor [Ac-DEVD-CHO, 10 µM, Sigma], for 1 h and then infected with A. hydrophila as mentioned earlier (16 (link)). The inhibitor concentrations used in the study had no adverse effects on the viability of HKM and bacterial growth per se (data not shown).
8
Apoptosis Induction in Glioma Cells
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The SW used in this study was extracted from Oxytropis kansuensis Bunge and characterized in our laboratory according to the published protocol 22 . Its purity was up to 99%. SW was dissolved in phosphate buffered saline (PBS, 0.01 mol/L, pH 7.2) at 10 mg/mL as stock solution, and stored at -20 oC after sterile filtration.
Antibodies against caspase-8, caspase-9, caspase-3, PARP, Bid, Bcl-2, Bax, cytochrome c, COX IV and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Wuhan Boster Bio-Engineering (Wuhan, China). Caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) were all purchased from Sigma-Aldrich (St. Louis, MO, US). TUNEL BrightRed Apoptosis Detection Kit was purchased from Vazyme (NJ, USA). All of other chemicals and reagents were the highest quality and obtained from standard commercial sources.
Primary GTCs were obtained according to our previous study 23 (link), and cultured in complete DMEM/F12 culture medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin, at 37 oC in a 5% CO2 atmosphere incubator. All animal experiments were carried out in accordance with policy and ethical guidelines.
Antibodies against caspase-8, caspase-9, caspase-3, PARP, Bid, Bcl-2, Bax, cytochrome c, COX IV and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Wuhan Boster Bio-Engineering (Wuhan, China). Caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) were all purchased from Sigma-Aldrich (St. Louis, MO, US). TUNEL BrightRed Apoptosis Detection Kit was purchased from Vazyme (NJ, USA). All of other chemicals and reagents were the highest quality and obtained from standard commercial sources.
Primary GTCs were obtained according to our previous study 23 (link), and cultured in complete DMEM/F12 culture medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin, at 37 oC in a 5% CO2 atmosphere incubator. All animal experiments were carried out in accordance with policy and ethical guidelines.
9
Apoptosis Signaling Pathway Analysis
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ATR-1, Z-DEVD-fmk (caspase-3 inhibitor), Z-LEHD-fmk (caspase-9 inhibitor), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against AKT, p-AKT, and PI3K were from Abcam Inc. (Cambridge, UK). Antibodies against CDK1, cyclin B1, Bax (6A7), mTOR, PTEN and p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against caspase-9, caspase-3, Bcl-2, Mcl-1, Bcl-xl, Bad, Bax, Bak, Cytochrome-c, Smac/Diablo, Cox-IV, cdc25c, p-Cdc25cand β-actin were from Cellular Signaling Technology (Danvers, MA, USA).
10
In Vitro Cytotoxicity Screening of Cancer Cells
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The cervical cancer (HeLa), ovarian cancer (OVCAR-3), breast cancer (MCF-7), lung cancer (A549) and prostate cancer (PC-3) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Flasks and 96-well plates for cell culture were purchased from Corning Inc. (Corning, NY, USA). The Dulbecco's modified Eagle's medium (DMEM) was obtained from Thermo Fisher Scientific Inc., (Gibco; Waltham, MA, USA) and the new-born calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Cell Counting kit-8 (CCK-8) was bought from Dojindo Molecular Technologies Inc. (Kumamoto, Japan). The DHA, DOX, Hoechst 33258 dye, broad-spectrum caspase inhibitors Z-IETD-fmk, Z-LEHD-fmk and Z-DQMD-fmk, and staurosporin (STS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluoroscein isothiocyanate (FITC)-Annexin V/propidium iodide (PI) apoptosis kit was purchased from eBioscience Inc. (San Diego, CA, USA).
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