For cell viability assay, cells were seeded in black 96-well plates with 2×103 cells per well in triplicate and allowed to attach overnight. Cells were treated with DMSO, celastrol, Z-VAD-FMK (MedChem Express, Shanghai, China), or both celastrol and Z-VAD-FMK at the indicated concentrations for the indicated time. According to the manufacturer’s protocol, the cell proliferation was measured by ATPlite luminescence assay (PerkinElmer, Norwalk, CT, United States) at the end of the incubation. The IC50 values were measured by the Logit method.
Z vad fmk
Manufactured by MedChemExpress
Sourced in United States, China
Z-VAD-FMK is a broad-spectrum caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes. It is commonly used as a research tool to study the role of apoptosis in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Ferrostatin 1, by MedChemExpress (13 mentions)
Necrostatin 1, by MedChemExpress (11 mentions)
Erastin, by MedChemExpress (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Fer 1, by MedChemExpress (7 mentions)
3 methyladenine, by MedChemExpress (6 mentions)
Necrosulfonamide, by MedChemExpress (6 mentions)
Mg132, by MedChemExpress (6 mentions)
3 methyladenine 3 ma, by MedChemExpress (5 mentions)
Nec 1, by MedChemExpress (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Ferrostatin 1, by Selleck Chemicals (4 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Cycloheximide (chx), by MedChemExpress (4 mentions)
Z devd fmk, by MedChemExpress (3 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Deferoxamine, by MedChemExpress (3 mentions)
Sulfasalazine, by MedChemExpress (3 mentions)
Liproxstatin 1, by Selleck Chemicals (3 mentions)
96 protocols using z vad fmk
1
Cell Viability Assay Protocol
2
Evaluating Oxidative Stress in Pancreatic Cancer
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Pancreatic cancer cells were incubated for 72 h in 60 mm
2 culture dishes with glutamine-free medium, medium containing 2 mM glutamine, or medium containing 2 mM glutamine supplemented with 50 μM diazooxonorleucine (Cat. #HY-108357; MedChemExpress, Shanghai, China) or 10 μM azaserine (Aza; Cat. #HY-B0919; MedChemExpress). Then, the cells were incubated with 10 μM 2’,7’-DCFH-DA (Cat. #D6883; Sigma-Aldrich, St Louis, USA) in serum-free DMEM at 37°C for 30 min. Cells treated with 100 μM H
2O
2 were used as positive controls. Thereafter, cells were treated with glutamine-free medium containing 2 μM ferrostatin-1 (Cat. #HY-100579; MedChemExpress), 5 mM N-acetylcysteine (NAC, Cat. #HY-B0215; MedChemExpress), 50 μM Z-VAD-FMK (Cat. #HY-16658B; MedChemExpress), and 150 μM necrostatin-1 (Cat. #HY-15760; MedChemExpress) to counteract cell death. The cells were then washed twice, resuspended, and filtered into single-cell suspensions before analysis. The fluorescence intensity of DCFH, formed by the reaction of DCFH-DA dyes with ROS, was measured at excitation and emission wavelengths of 488 and 530 nm, respectively, with a FC500 MPL flow cytometer (Beckman Coulter, Pasadena, USA). A minimum of 10,000 cells were analyzed per condition
[13] (link). Proportion analysis and histogram generation were performed using FlowJo software (version 10.6; FlowJo, Ashland, USA).
2 culture dishes with glutamine-free medium, medium containing 2 mM glutamine, or medium containing 2 mM glutamine supplemented with 50 μM diazooxonorleucine (Cat. #HY-108357; MedChemExpress, Shanghai, China) or 10 μM azaserine (Aza; Cat. #HY-B0919; MedChemExpress). Then, the cells were incubated with 10 μM 2’,7’-DCFH-DA (Cat. #D6883; Sigma-Aldrich, St Louis, USA) in serum-free DMEM at 37°C for 30 min. Cells treated with 100 μM H
2O
2 were used as positive controls. Thereafter, cells were treated with glutamine-free medium containing 2 μM ferrostatin-1 (Cat. #HY-100579; MedChemExpress), 5 mM N-acetylcysteine (NAC, Cat. #HY-B0215; MedChemExpress), 50 μM Z-VAD-FMK (Cat. #HY-16658B; MedChemExpress), and 150 μM necrostatin-1 (Cat. #HY-15760; MedChemExpress) to counteract cell death. The cells were then washed twice, resuspended, and filtered into single-cell suspensions before analysis. The fluorescence intensity of DCFH, formed by the reaction of DCFH-DA dyes with ROS, was measured at excitation and emission wavelengths of 488 and 530 nm, respectively, with a FC500 MPL flow cytometer (Beckman Coulter, Pasadena, USA). A minimum of 10,000 cells were analyzed per condition
[13] (link). Proportion analysis and histogram generation were performed using FlowJo software (version 10.6; FlowJo, Ashland, USA).
3
Comprehensive Compound Acquisition Protocol
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Sorafenib (#T0093L) was purchased from TargetMol (Shanghai, China); lenvatinib (#S1164), brivanib (#S1084) and bafilomycin A1 (#S1413) were purchased from Selleck (Shanghai, China); cisplatin (#P4394), doxorubicin (#D1515), N-acetyl-l -cysteine (NAC, #A7250), and Hoechst (#94403) were purchased from Sigma‒Aldrich (Shanghai, China); canagliflozin (#A11100) was purchased from AdooQ Bioscience (Nanjing, China); and oligomycin (#HY-N6782), antimycin A (#HY-105755), phloretin (#HY-N0142), z-VAD-FMK (#HY-16658B), necrostatin-1 (#HY-15760), and ferrostatin-1 (#HY-100579) were purchased from MedChemExpress (Shanghai, China).
4
Autophagy and Apoptosis Inhibition in PANC-1 Cells
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The PANC-1 cells were seeded into 35-mm-diameter culture dishes (2 × 105 cells/dish) and allowed to adhere overnight. For the autophagy inhibitor analysis, the cells were pretreated with 1 mM 3-Methyladenine (3-MA) (MedChem Express, Monmouth Junction, NJ, USA) for 1 h. For the apoptosis inhibitor analysis, the cells were pretreated with 20 μM Z-VAD-FMK (MedChem Express) for 1 h.
5
Investigating NSCLC Cell Line Responses
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Human NSCLC cell HCC827, HCC1975, H1650, and H460 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO2. All cells were maintained at the incubator according to the standard protocols and subjected to routinely checking for mycoplasma contamination. Antibodies against HK2 (#2867), Akt (#4691), p-Akt (#4060), cleaved-caspase 3 (#9664), Bax (#14796), VDAC1 (#4661), α-tubulin (#2144), cytochrome c (#4280), Akt1 (#2938), Akt2 (#2964), p-GSK3β (#5558), β-actin (#3700), anti-rabbit IgG HRP (#7074), and anti-mouse IgG HRP (#7076) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). The natural compound Erianin was from Selleck Chemicals (Houston, TX). Necrostatin-1, z-VAD-fmk, and 3-methyladenine were purchased from MedChemExpress (New Jersey, US). Lipofectamine 2000 transfection reagent for transient transfection was purchased from Thermo Fisher Scientific (Waltham, MA).
6
Osthole and Cisplatin Co-treatment Induces Apoptosis
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Osthole and cisplatin were purchased from Meilunbio (purity≥98%, Dalian, China). Dicoumarol (DIC), Z-VAD-FMK, Z-DEVD-FMK and 3-Methyladenine (3-MA) were purchased from MedChem Express (Princeton, NJ, USA). N-Acetyl-L-cysteine (NAC) and JC-1 were purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) and 2′, 7′-Dichlor-o uorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-rabbit IgG, anti-mouse IgG and antibodies against β-actin were purchased from ZSGB-BIO (Beijing, China). Anti-LC3A/B antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NQO1, anti-caspase-3, anti-cleaved caspase-3, anti-PARP-1 and anti-GSDME antibodies were purchased from Abcam (Cambridge, MA, USA). FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, CA, USA)
7
Establishing Drug-Resistant Hepatocellular Carcinoma Cell Lines
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A normal hepatocyte line (L02) and HCC cell lines (Hep3B, HepG2, Huh7, and PLC) were purchased from Shanghai Institute of Cell Bank (Shanghai, China). Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with NaHCO3 (1.5 g/L). HepG2 and PLC cells were grown in minimum essential medium (Gibco, Grand Island, USA) with NaHCO3 (1.5 g/L) and sodium pyruvate. Cells were cultured with 10% fetal bovine serum (Pansera ES, Pan biotech GmbH, Germany), penicillin (U/ml), and streptomycin (0.1 mg/ml) at 37°C in a humidified atmosphere containing 5% CO2. Cells were exposed to sorafenib (Solarbio, Beijing, China) for the indicated time and at the indicated concentration. Drug-resistant cell lines were established by stepwise selection of cells cultured in growth media with increasing concentrations of the drug over a period of 6 months. Erastin, staurosporine (STS), ferrostatin-1 (Fer-1), deferoxamine (DFO), ZVAD-FMK, and necrosulfonamide (NSA) were purchased from MedChemExpress (New Jersey, USA). sorafenib and pioglitazone (Pg) were purchased from Solarbio Biotechnology Company (Beijing, China).
8
Transfection of HeLa Cells with Z-VAD-FMK
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HeLa cells obtained from the Department of Medicine, Jinan University (Guangzhou, China) were cultured just as described previously 16 . When cells reached 70–90% confluence in a 35‐mm glass dish, cells were transfected with indicated amounts of plasmids in the presence or absence of 50 µm Z‐VAD‐FMK (MedChem Express) using TurboFect Transfection Reagent (Thermo Scientific, Waltham, MA, USA) for 24 h.
9
Culturing Liver Cancer Cell Lines
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Human liver cancer cells HepG2 were obtained from the American Type Culture Collection (ATCC). HCCC9810 was obtained from Procell (Catalog, CL-0095). Cell cultures were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (EallBio, Beijing, China; # 03. U16001) at 37°C in a humidified atmosphere of 95% air and 5% CO2. RSL3, dimethyl sulfoxide (DMSO), Z-VAD-FMK, necrosulfonamide, and ferrostatin-1 were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).
10
Macrophage Ferroptosis Induction Assay
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Macrophages were implanted in a 96-well plate and cultured for 24 h at 37 °C. After macrophages had undergone different treatments at specific times, the supernatant was removed, and then the cells were washed with PBS three times. 100 μL 1640 medium containing 10 μL Cell Counting Kit-8 (CCK-8, Byotime, Beijing, China) was added into each well. The cells were incubated for 1 h at 37 °C until they turned orange and then measured at an absorbance of 450 nm. All drugs and corresponding treatment concentration: erastin (HY-15763), 10 μM; RSL3 (HY-100218A), 5 μM Ferrostatin-1 (HY-100579), 10 μM; deferoxamine (HY-B0988), 200 μM; necrostatin (HY-15760) 2 μM; ZVAD-FMK(HY-16658B), 5 μM were purchased from MedChemExpress (MCE). FeSO4 7H2O (F7002), 200 μg/mL; baicalein (465119), 20 μM were purchased from Sigma.
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