Jurkat, Molt4, CCRF-CEM, SupT1 and U1 cells were maintained in RPMI1640 medium (GIBCO) with 10% fetal bovine serum (FBS, GIBCO), and antibiotics (GIBCO). For exogenous stimulation, the cells were treated with PMA (LC Laboratories), Ionomycin (CALBIOCHEM), or TNF-α (R&D systems). For inhibition of epigenetic factors, the cells were treated with the indicated concentrations of VPA (SIGMA), SAHA (Cayman), TSA (SIGMA), 3-deazaneplanocin A (DZNep, Cayman), or GSK126 (Chemie Tek). HEK293T, HEK293FT, and Hela/LTR-luciferase cells were maintained in DMEM with 10% FBS and antibiotics. Human peripheral blood mononuclear cells (PBMCs) were prepared from whole blood of healthy donors by density gradient centrifugation with Ficoll-Paque (GE healthcare). CD4+ T cells were purified by CD4+ T cell isolation kit (Miltenyi Biotec) and maintained in RPMI1640 with 10% of FBS. T cell activation was accomplished by treating the anti-CD3/CD28 antibodies (Miltenyi Biotec).
3 deazaneplanocin a
Manufactured by Cayman Chemical
Sourced in United States
3-Deazaneplanocin A is a chemical compound that can be used as a laboratory reagent. It is a synthetic analog of the natural product neplanocin A. The core function of 3-Deazaneplanocin A is as a chemical tool for research purposes, but no further details on its intended use can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
A2780, by National Collection of Authenticated Cell Cultures (2 mentions)
Gsk126, by Cayman Chemical (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Dznep, by Cayman Chemical (1 mentions)
Gsk126, by Chemietek (1 mentions)
Anti cd3 cd28 antibodies, by Miltenyi Biotec (1 mentions)
Phorbol myristate acetate (pma), by LC Laboratories (1 mentions)
Skov3, by National Collection of Authenticated Cell Cultures (1 mentions)
Scrambled shrna, by Addgene (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
5 azacytidine, by Merck Group (1 mentions)
Caccinh a01, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Cycloheximide (chx), by Cayman Chemical (1 mentions)
7 protocols using 3 deazaneplanocin a
1
Cell culture and treatment protocol
2
Ovarian Cancer Cell Lines and EZH2 Modulation
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The human ovarian cancer cell lines A2780, SKOV3 and ES2 were obtained from the China Center for Type Culture Collection (China). SKOV3LV-EZH2 was constructed by lentivirus with upregulated EZH2, while SKOV3LV-NC was the control. A2780sg87 and SKOV3sg87 were obtained by CRISPR/Cas9 lentivirus transfection targeting inhibition of EZH2, SKOV3 sg87-13# was obtained from monoclonal selection using the infinite dilution method, and SKOV3sgnc, A2780sgnc were the corresponding control. A2780sh950 was obtained by shRNA targeting EZH2, and A2780shnc was the control. Cells were maintained in a humidified incubator with 5% CO2 (37°C) in DMEM supplemented with 10% FBS. 3-Deazaneplanocin A (DZNep) and GSK126 were purchased from Cayman Chemical, USA.
3
Modulating Epigenetic Pathways in Cell Studies
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For in vitro drug studies, SAHA and Trichostatin A (Sigma-Aldrich) were dissolved in DMSO, and diluted in media to final concentrations of 500 nM–2 μM and 250–15.6 nM, respectively. Nicotinamide (Sigma-Aldrich) was dissolved in ddH2O and diluted in media to 5 mM concentration. 5-azacytidine (Sigma-Aldrich) and 3-Deazaneplanocin A (Cayman Chemicals) were dissolved in DMSO and diluted in media to final concentration of 5 μM. CaCCInh-A01 (Sigma-Aldrich) was dissolved in DMSO and diluted in media to a final concentration of 10 μM. Every 48 h, media with fresh drug was changed during each experiment.
For short hairpin RNA (shRNA)-mediated knockdown, the PLKO.1 background vector was used (GE Life Sciences, Mickleton, NJ, USA). shRNA plasmids were packaged using a third-generation lentiviral system (pMDL, VSV-G and pRSV-REV), and expressed in HEK-293T cells. Human EPAS1 TRCN: 0000003805, 0000003807; Mouse EPAS1 TRCN: 0000082306, 0000082307. Mouse ANO1 TRCN: 0000173335, 0000193423. Scrambled shRNA was obtained from Addgene (Cambridge, MA, USA). Viral supernatant was collected 24 and 48 h post-transfection, and concentrated using 10-kDA Amicon Ultra-15 centrifugal filter units (Millipore, Billerica, MA, USA). For re-expression studies, human HIF-2α (ref. 18 (link)) open reading frame was sub-cloned into the pCDH-CMV-MCS-EF1-PURO mammalian expression vector.
For short hairpin RNA (shRNA)-mediated knockdown, the PLKO.1 background vector was used (GE Life Sciences, Mickleton, NJ, USA). shRNA plasmids were packaged using a third-generation lentiviral system (pMDL, VSV-G and pRSV-REV), and expressed in HEK-293T cells. Human EPAS1 TRCN: 0000003805, 0000003807; Mouse EPAS1 TRCN: 0000082306, 0000082307. Mouse ANO1 TRCN: 0000173335, 0000193423. Scrambled shRNA was obtained from Addgene (Cambridge, MA, USA). Viral supernatant was collected 24 and 48 h post-transfection, and concentrated using 10-kDA Amicon Ultra-15 centrifugal filter units (Millipore, Billerica, MA, USA). For re-expression studies, human HIF-2α (ref. 18 (link)) open reading frame was sub-cloned into the pCDH-CMV-MCS-EF1-PURO mammalian expression vector.
4
Establishing Cisplatin-Resistant Ovarian Cancer Cells
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The A2780 and ES2 human ovarian cancer cell lines were purchased from the China Center for Type Culture Collection (China) and validated by short tandem repeat profiling (Biowing Applied Biotechnology, Shanghai, China). The cDDP‐resistant cell lines A2780C12 and ES2C12 were generated by exposing the parent cells to cDDP with increasing drug concentrations for 12 cycles. Cells were grown in DMEM supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37°C. Cisplatin was purchased from Sigma (USA). BODIPY‐Pt was a gift from Dr. Wenliang Li (National Engineering Laboratory for Druggable Gene and Protein Screening, School of Life Science, Northeast Normal University, Changchun, China). 3‐Deazaneplanocin A (DZNEP), GSK126, cycloheximide (CHX), and MG132 were purchased from Cayman Chemical, USA.
5
RA and DZNep Treatment Protocol
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All trans-RA was bought from Sigma (St Louis, MO, USA, Sigma R2625-50 mg). RA was dissolved in ethanol to make a 5 mM stock. 3-Deazaneplanocin A (DZNep), an EZH2 inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA). DZNep was dissolved in dimethyl sulfoxide (DMSO) and stored at 5 mM concentration.
6
Modulation of Cellular Stress Responses
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Hydrogen peroxide (H2O2) was purchased from Sigma (H1009). 3-Deazaneplanocin-A (DZNep) was purchased from Cayman Chemicals (13828). ATM inhibitor was purchased from Tocris (KU55933), Epigallocatechin-Gallate (ECGC) was purchased from Calbiochem (324880), N-acetyl-L-Cysteine (NAC) and IKK-β inhibitor (TPCA-1) were purchased from Sigma (A9165-5gr and T1452).
Concentrations used were: H2O2 (100 & 200 µM), DZNep: (0.5 µM), KU55933: (10 µM), ECGC: (10 µM), NAC: (5 mM), TPCA-1: (10 µM).
Lymphotoxin β receptor agonist antibody was used at a final concentration used of 2 µG/mL.
Concentrations used were: H2O2 (100 & 200 µM), DZNep: (0.5 µM), KU55933: (10 µM), ECGC: (10 µM), NAC: (5 mM), TPCA-1: (10 µM).
Lymphotoxin β receptor agonist antibody was used at a final concentration used of 2 µG/mL.
7
Combinatorial Treatment of Leukemia Cells
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Cell samples were treated with 1 μM Retinoic acid (Sigma-Aldrich, St. Louis, MO, USA), 2 nM or 8 nM Idarubicin (Sigma-Aldrich), 0.5 μM 3-Deazaneplanocin A (Cayman Chemical Company, Ann Arbour, MI, USA), and 0.2 μM Belinostat (PXD101) (Selleckchem, Munich, Germany) in different combinations. Cell proliferation and survival were evaluated by trypan blue exclusion test. Cells were mixed with 0.2% of trypan blue dye (final concentration). Viable and dead (blue coloured) cell numbers were determined by counting the cells in a haemocytometer under the light microscope. For the detection of apoptosis, we used the assay “ApoFlowEx® FITC Kit” (Exbio, Vestec, Czech Republic) according to the manufacturer's instructions. This assay detects viable, early apoptotic, and late apoptotic or necrotic cells according to how they get stained by Annexin V-FITC and Propidium Iodide. Stained cells were analysed on the BD FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle distribution was analysed using standard propidium iodide staining procedure [15 (link)].
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