Z vad fmk

Manufactured by Adooq

Sourced in United States

Z-VAD-FMK is a broad-spectrum caspase inhibitor used for research purposes. It functions by blocking the activity of various caspase enzymes, which play a role in the apoptosis (programmed cell death) pathway.

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Lab products found in correlation

Cellrox green, by Thermo Fisher Scientific (2 mentions) Marimastat, by Santa Cruz Biotechnology (2 mentions) Cytotox green, by Sartorius (2 mentions) Mg 132, by Promega (2 mentions) Px ethyl, by Merck Group (2 mentions) Cy and alexa fluor conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions) Abt 263, by Adooq (2 mentions) Mk 2206, by Adooq (2 mentions) Ly294002, by Adooq (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Hydroxychloroquine, by Cayman Chemical (1 mentions) Lapatinib, by Cayman Chemical (1 mentions) Staurosporine, by Fujifilm (1 mentions) N acetyl l cysteine, by Fujifilm (1 mentions) Gefitinib, by Cayman Chemical (1 mentions) Abemaciclib, by Adooq (1 mentions) Pepstatin a, by Peptide Institute (1 mentions) Bafilomycin a1, by Fujifilm (1 mentions) Hoechst 33342, by Nacalai Tesque (1 mentions) Hank s balanced salt solution, by Fujifilm (1 mentions)

19 protocols using z vad fmk

Cathepsin B Inhibition Assay

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The assay was performed essentially as described by Mendieta et al.^{60 (link)} with Cathepsin B (human, recombinant, active - BioVision #7580-5), Substrate Z-Arg-Arg-AMC (Sigma C5429) and 1 µM Z-VAD-FMK (Adooq #A12373) as positive inhibition control.
The assay was maintained at 37 °C and monitored every 2 min for 16 min at 348 nm/440 nm (excitation/emission).

Bosc D., Vezenkov L., Bortnik S., An J., Xu J., Choutka C., Hannigan A.M., Kovacic S., Loo S., Clark P.G., Chen G., Guay-Ross R.N., Yang K., Dragowska W.H., Zhang F., Go N.E., Leung A., Honson N.S., Pfeifer T.A., Gleave M., Bally M., Jones S.J., Gorski S.M, & Young R.N. (2018). A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B. Scientific Reports, 8, 11653.

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Inhibitor Compounds for Cell Death Pathways

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Lapatinib, FTY720, hydroxychloroquine (HCQ), U18666A and gefitinib were purchased from Cayman Chemical Company and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM to obtain stock solutions. Abemaciclib and z-VAD-FMK, pan-caspase inhibitors, were purchased from AdooQ BioScience. E64d and pepstatin A, inhibitors of lysosomal proteases, were purchased from Peptide Institute, Inc. Necrostatin-1, a specific inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), was purchased from Enzo Life Sciences, Inc., and N-acetyl-L-cysteine, staurosporine, bafilomycin A₁ and Hanks' balanced salt solution (HBSS) were obtained from FUJIFILM Wako Pure Chemical Corporation. Hoechst 33342 was obtained from Nacalai Tesque, Inc., and DAPI was purchased from Sigma-Aldrich (Merck KGaA).

Suzuki S., Ogawa M., Miyazaki M., Ota K., Kazama H., Hirota A., Takano N., Hiramoto M, & Miyazawa K. (2021). Lysosome-targeted drug combination induces multiple organelle dysfunctions and non-canonical death in pancreatic cancer cells. Oncology Reports, 47(2), 40.

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Immunocytochemistry with VE-cadherin antibody

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Mouse anti-VE-cadherin antibody (sc-6458) diluted 1:50 was obtained from Santa Cruz Biotechnology (United States). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (United States) and Molecular Probes (United States), respectively, and used for immunocytochemistry. Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, United States) and Santa Cruz Biotechnology (sc-3067, United States). Marimastat from Santa Cruz Biotechnology (sc-202223, United States), MG-132 from Promega (G9951, United States). Cytotoxgreen was obtained from Essen BioScience (United States) and CellROXgreen from Molecular Probes (United States). PX-ethyl was purchased from Sigma (United States), according to Sigma safety data sheet safety measures of eyeshields, face shields, full-face respirator and Gloves should be taken. For assessing the BBB response, PX freshly made in ethanol to 400 mM stock solution was immediately diluted in the medium to the desired final concentrations and added to the cell culture. All other reagents applied in this study were used in accordance to the known literature and the supplier's guidelines.

Rand D., Ravid O., Atrakchi D., Israelov H., Bresler Y., Shemesh C., Omesi L., Liraz‐Zaltsman S., Gosselet F., Maskrey T.S., Beeri M.S., Wipf P., & Cooper I. (2020). Endothelial iron homeostasis regulates BBB integrity via the HIF2α – Ve-cadherin pathway.

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Lymphoma Cell Lines Treated with ROS Scavengers

Cited 1 time

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The lymphoma cell lines Jurkat (ACC282) and U-937 (ACC5) were used. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Corning, Wiesbaden, Germany) containing 10% fetal bovine serum (Sigma-Aldrich, Taufkirchen, Germany), 1% glutamine (Corning, Wiesbaden, Germany), and 1% penicillin/streptomycin (Corning, Wiesbaden, Germany) at 37 °C and 5% CO₂. At 7.5 × 10⁵ per 1.5 mL of medium, the cells were seeded into square Petri dishes having 25 compartments, each with a surface area of 3.7 cm² (Thermo Scientific, Zurich, Switzerland), directly before the treatment. Catalase (cat, 20 µg/mL; Sigma-Aldrich, Taufkirchen, Germany) or N-acetylcysteine (NAC, 2 mM; Sigma-Aldrich, Taufkirchen, Germany) were used as the ROS scavenger. The following inhibitors were used to interfere with apoptosis or ferroptosis signaling pathways: Z-VAD-FMK (50 µM; AdooQ Bioscience, Irvine, CA, USA), a pan-caspase inhibitor, and liproxstatin-1 (50 nM; Sigma-Aldrich, Taufkirchen, Germany), an inhibitor of lipid peroxidation [50 (link)].

Wolff C.M., Kolb J.F., Weltmann K.D., von Woedtke T, & Bekeschus S. (2020). Combination Treatment with Cold Physical Plasma and Pulsed Electric Fields Augments ROS Production and Cytotoxicity in Lymphoma. Cancers, 12(4), 845.

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Measuring 14-3-3 Degradation in Cells

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Lysates of SHSY5Y cells or zebrafish brain tissue (5 mg protein) were separated in 10% TGX gels (Bio-Rad) and transferred onto PVDF membrane (Bio-Rad) using the Transblot system (Bio-Rad). Western blotting membranes were incubated with rabbit anti-pan-14-3-3 (sc-629, RRID: AB_2273154; 1:1000; Santa Cruz Biotechnology) as primary antibody. Mouse anti-b-actin (A1978, RRID: AB_476692; 1:1000; Sigma-Aldrich) or rabbit anti-GAPDH (ab9485, RRID: AB_307275;1:1000; Abcam) was used as loading control, and goat anti-rabbit (170-6515, RRID: AB_11125142; 1:1000; Bio-Rad) and goat anti-mouse (170-6516, RRID: AB_11125547; 1:1000; Bio-Rad) were used as secondary antibodies. Ebselen-treated samples were referenced to untreated, which were given the arbitrary value of 1.
For experiments on 14-3-3 degradation in cells, 500,000 SH-SY5Y cells per well were seeded in six-well plates; 5 hours postseeding, cells were treated with 5-10 mM ebselen or ebselen oxide and 50 mM Z-VAD-FMK (caspase inhibitor; Adooq Biosciences) and DMSO (0.05%) as a control (without ebselen and caspase inhibitor). Cells were incubated for 16 hours before treating with 500 nM bortezomib (proteasome inhibitor; Fisher Scientific) for 2 hours before collecting for Western blot analysis. Densitometric analysis was performed using Image Laboratory Software 6.0.1 (Bio-Rad).

Waløen K., Jung-Kc K., Vecchia E.D., Pandey S., Gasparik N., Døskeland A., Patil S., Kleppe R., Hritz J., Norton W.H.J., Martinez A, & Haavik J. (2021). Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins. Molecular pharmacology, 100(2).

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Murine Fibroblast Infection Assay

Cited 2 times

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Mouse L929 fibroblasts were originally obtained from the American Type Culture Collection (ATCC) and were grown in Eagle’s minimal essential medium (EMEM, Wisent) containing 5% fetal bovine serum (Wisent) and supplemented with 1% glutamine. MRV serotype 3 strain Dearing (T3/Human/Ohio/Dearing/55) was also originally obtained from ATCC and was propagated and titrated by TCID₅₀ on L929 fibroblasts [25 (link)]. The laboratory stock of MRV type 3 (T3D^S) was previously described [26 (link),27 (link)], and rescued by reverse genetics following the introduction of the appropriate mutations in the plasmids encoding the virus from the original reverse genetics system [28 (link)]. The pan-caspase inhibitor zVAD-fmk (AdooQ Bioscience), the RIPK3 inhibitor GSK872 (Abcam) and the RIPK1 inhibitor GSK963 (Sigma) were resuspended at 10 mM in DMSO and used at various final concentrations. To elicit RIPK1-mediated necroptosis in L929 cells, cells were pretreated with zVAD-fmk at 100 μM for 1 h and then recombinant murine TNF-α (Peprotech) was directly added to the medium at 25 ng/mL. Recombinant mouse interferon-β (PBL Assay Science, #12401-1) was diluted in complete medium to the indicated concentration and cells were treated for 5 h.

Boudreault S., Lemay G, & Bisaillon M. (2022). U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction. Viruses, 14(12), 2710.

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Maintenance and Calcium Signaling in PC3 and LNCaP Cells

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PC3 cells (ATCC, CRL-1435) and LNCaP cells (ATCC, CRL-1740) were maintained in RPMI 1640 (Thermo Fisher Scientific, 21875091) containing 10% FBS (Sigma, F7524 batch BCBT0730), and cultured at 37°C in a humidified 5% CO2 incubator. In experiments with EGTA, cells were incubated in calcium-free DMEM (Thermo Fisher Scientific, 21068028) with 10% FBS. Thapsigargin epoxide (EpoTg) was prepared as described previously [40] . Other compounds were: Thapsigargin (Sigma, T9033), A23187 (Sigma, T9033), ATP (Sigma, A6419), Ionomycin (Sigma, I9657), Bafilomycin A1 (Enzo, BML-CM110), EGTA (Sigma, E8145), Z-VAD-FMK (Adooq Bioscience, A12373), and Propidium Iodide (Merck, 537059).

Szalai P., Parys J.B., Bultynck G., Christensen S.B., Nissen P., Møller J.V, & Engedal N. (2018). Nonlinear relationship between ER Ca²⁺ depletion versus induction of the unfolded protein response, autophagy inhibition, and cell death. Cell calcium, 76.

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Apoptosis Inhibition During CV-B5 Infection

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Cells were pretreated with serum-free medium containing 100 μM z-VAD-FMK (Adooq) and then exposed to CV-B5/F for an additional 48 h.

Cui B., Song L., Wang Q., Li K., He Q., Wu X., Gao F., Liu M., An C., Gao Q., Hu C., Hao X., Dong F., Zhou J., Liu D., Song Z., Yan X., Zhang J., Bai Y., Mao Q., Yang X, & Liang Z. (2023). Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors. Signal Transduction and Targeted Therapy, 8, 366.

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Chemical Treatments for Cell Studies

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Z-VAD-FMK, chloroquine (CQ), Rapamycin (Rapa), NU7441, Cyt387, A674563, KU60019, LY294002, PD0332991, AT7519, MK2206, CUDC907, LY2109761, GANT61, BIX 02189, Spautin-1, QNZ, LY317615, PD169316, GSK2606414, LYK974, TCK ERK 11e, SC79, MC1568, H89, ICG001, Perifosine, AEE788, ABT263, GDC-0941, FH535, PD0325025, NU7026, STF-083010, AEBSF HCl were purchased from Adooq. N-Acetyl-l-cysteine ethyl ester, DMSO, PEG300, and Tween-80 were purchased from MCE.

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Immunocytochemistry Reagents and Protocols

Cited 3 times

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Mouse anti-VE-cadherin antibody (sc-9989) diluted 1:50 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (Philadelphia, PA, USA) and Molecular Probes (Eugene, OR, USA), respectively, and used for immunocytochemistry. HIF2α inhibitor [51 (link)] (Axon 2034) was obtained from Axon Medchem (Hanzeplein, The Netherlands). Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, Irvine, CA, USA) and Santa Cruz Biotechnology (sc-3067, Dallas, TX, USA). Marimastat from Santa Cruz Biotechnology (sc-202223, Dallas, TX, USA), MG-132 from Promega (G9951, Madison, WI, USA). Cytotoxgreen was obtained from Essen BioScience (Ann Arbor, MI, USA) and CellROXgreen from Molecular Probes (Eugene, OR, USA). PX-ethyl was purchased from Sigma (St. Louis, MO, USA). According to the Sigma safety data sheet, safety measures of eye shields, face shields, full-face respirator, and gloves should be taken. PX was used according to our previously reported protocol [43 (link),44 (link)] All other reagents applied in this study were used in accordance to the known literature and the supplier’s guidelines.

Rand D., Ravid O., Atrakchi D., Israelov H., Bresler Y., Shemesh C., Omesi L., Liraz-Zaltsman S., Gosselet F., Maskrey T.S., Schnaider Beeri M., Wipf P, & Cooper I. (2021). Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α—Ve-Cadherin Pathway. Pharmaceutics, 13(3), 311.

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