Z vad ome fmk

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Z-VAD(OMe)-FMK is a broad-spectrum irreversible caspase inhibitor. It functions by binding to the catalytic site of caspases, preventing their activation and subsequent cellular apoptosis.

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Z-VAD-FMK (56 citations) Z-VAD (12 citations)

5 protocols using z vad ome fmk

TP4 Peptide Synthesis and Characterization

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TP4 (FIHHIIGGLFSAGKAIHRLIRRRRR) were synthesized and purified by GL Biochem Ltd. (Shanghai, China) as previously described [27 (link)]. Necrosis inhibitor, Necrox-2, and pan-Caspase inhibitor, Z-VAD(OMe)-FMK, were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) N-(3-Chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (Gefitinib) and N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine hydrochloride (Erlotinib) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Primary antibodies were purchased from the Cell Signaling (Danvers, MA, USA) (Lamin A/C, clone 4C11; Lamin B1, clone D4Q4Z; Caspase 3; cleaved Caspase 3) and EMD Millipore (GAPDH, clone 6C5) (Temecula, CA, USA). The Structural model and the helical projection of TP4 were generated, as previously described [41 (link)].

Ting C.H, & Chen J.Y. (2018). Nile Tilapia Derived TP4 Shows Broad Cytotoxicity toward to Non-Small-Cell Lung Cancer Cells. Marine Drugs, 16(12), 506.

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Synthesis and Characterization of T315 Pyrazole Compound

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T315 {N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide} was synthesized as previously described [2 (link)], with identity and purity (≥99%) verified by proton nuclear magnetic resonance, high-resolution mass spectrometry, and elemental analysis. For in vitro experiments, T315 was dissolved in dimethyl sulfoxide (DMSO), and added to the culture medium with a final DMSO concentration less than 0.1%. The pharmacological agents were purchased from the respective vendors: bafilomycin-A1 (Cayman Chemical, Ann Arbor, MI, USA); chloroquine (Sigma-Aldrich, St. Louis, MO, USA); 3-methyladenine (3-MA; Sigma-Aldrich); Z-VAD(OMe)-FMK (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Chiu C.F., Weng J.R., Jadhav A., Wu C.Y., Sargeant A.M, & Bai L.Y. (2016). T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death. International Journal of Molecular Sciences, 17(8), 1337.

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McCoy Cell Apoptosis and Necroptosis Assay

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McCoy monolayers were treated with various concentrations of Z-VAD(OMe)-fmk (Santa Cruz Biotechnology), 50 µM necrostatin-1 (Santa Cruz Biotechnology), 100 µm of Z-DEVD-FMK, Z-IETD-FMK, or Z-LEHD-FMK (R&D Systems, Inc.), or dimethyl sulfoxide (DMSO) vehicle alone for 2 h prior to infection. Cells were infected at an MOI of 0.1, and IFN-γ was added in some cases. For inducer experiments, McCoy cells were infected at an MOI of 0.1 (EVI-H1 staining) or at an MO1 of 1 (acridine orange/ethidium bromide staining). At 20 hpi, 1 µM staurosporine (Santa Cruz Biotechnology), 5 ng/ml TNF-α (BioLegend-recombinant mouse) and 10 µg/ml CHX, or 5 ng/ml TNF-α and 10 µM ZVAD(OMe)-fmk was added. Cells were stained with acridine orange and ethidium bromide at 24 hpi or labeled with EVI-H1 as described above. To assay cell death, 4 µl of 100 µg/ml acridine orange (Sigma) and ethidium bromide (Sigma) were added to DMEM and incubated 15 min at room temperature (RT) (41 (link)). To assay inclusion lysis, cells were fixed with methanol, labeled with EVI-H1, and imaged as described above.

Giebel A.M., Hu S., Rajaram K., Finethy R., Toh E., Brothwell J.A., Morrison S.G., Suchland R.J., Stein B.D., Coers J., Morrison R.P, & Nelson D.E. (2019). Genetic Screen in Chlamydia muridarum Reveals Role for an Interferon-Induced Host Cell Death Program in Antimicrobial Inclusion Rupture. mBio, 10(2), e00385-19.

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Mitochondrial Dynamics and Apoptosis Assays

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Our experiments used the following reagents: Mono-2-ethylhexyl phthalate (MEHP, Sigma 796,832), Di-2-ethylhexyl phthalate (DEHP, Sigma 80,030), Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma C2759), N-Acetyl Cysteine (NAC, Merck), Z-VAD (OMe)-FMK (Santa Cruz, CAS 187389-52-2), Bafilomycin A1 (BafA1, Sigma B1793), MitoSOX™ (Invitrogen, M36008), MitoProbe™ DiIC₁(5) Assay Kit (Invitrogen, M34151), Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Abcam, ab113852), Propidium iodide (PI, Sigma P4170), Annexin V FITC (Invitrogen, V13242). We used the following antibodies: anti-phospho-DRP1 (Ser616) antibody (CST, 3455), anti-phospho-DRP1 (Ser637) antibody (CST, 4867), anti-DRP1 (D6C7) antibody (CST, 8570), anti-HSP60 (D6F1) antibody (CST, 12,165), anti-Parkin antibody (Santa Cruz, SC32282), anti-PINK1 (D8G3) antibody (CST, 6946), anti-Tim23 (H-8) antibody (Santa Cruz, SC514463), anti-Tom20 (FL-145) antibody (Santa Cruz, SC11415), anti-COXIV (3E11) antibody (CST, 4850), anti-Mitofusin-1 (D6E2S) antibody (CST, 14,739), anti-Mitofusin-2 (D1E9) antibody (CST, 11,925), anti-VDAC antibody (CST, 4866), anti-microtubule-associated protein 1 light chain 3/LC3 antibody (Sigma, L7543), anti-p62 antibody (Sigma, SAB1406748), anti-phospho-ubiquitin antibody (Merck Millipore, ABS1513-I), anti-ubiquitin (P4D1) antibody (Santa Cruz, SC8017), anti-β-actin antibody (Sigma, A5441).

Xu J., Wang L., Zhang L., Zheng F., Wang F., Leng J., Wang K., Héroux P., Shen H.M., Wu Y, & Xia D. (2020). Mono-2-ethylhexyl phthalate drives progression of PINK1-parkin-mediated mitophagy via increasing mitochondrial ROS to exacerbate cytotoxicity. Redox Biology, 38, 101776.

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Investigating Apoptosis and Autophagy Markers

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We used the following reagents in our experiments: Z-VAD (OMe)-FMK (Santa Cruz, CAS 187389-52-2), Chloroquine diphosphate (CQ, Sigma, C6628), N-Acetyl Cysteine (NAC, Merck), Propidium Iodide (PI, Sigma P4170), CM-H2DCFDA (Invitrogen, C6827). The antibodies in our experiments include: anti-PARP antibody (CST, 9542), anti-caspase-3 antibody (CST, 9662), anti-β-actin antibody (Sigma, A5441), anti-microtubule-associated protein 1 light chain 3/LC3 antibody (Sigma, L7543), anti-phospho-p70 S6 Kinase (Thr389) antibody (CST, 9205), anti-p70 S6 Kinase antibody (CST, 9202), anti-phospho-4E-BP1 (Ser65) antibody (CST, 2855 P), anti-4E-BP1 antibody (CST, 9644), anti-ATG7 antibody (CST, 2631), anti-p53 antibody (PharMingen, 554170), anti-p21 Wat 1/Cip1 (DCS60) antibody (CST, 2946).

Xu J., Wu Y., Lu G., Xie S., Ma Z., Chen Z., Shen H.M, & Xia D. (2017). Importance of ROS-mediated autophagy in determining apoptotic cell death induced by physapubescin B. Redox Biology, 12, 198-207.

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