5 azacytidine aza

Manufactured by Merck Group

Sourced in United States, Germany

5-Azacytidine (AZA) is a synthetic nucleoside analogue used in biochemical and cell biology research. It functions as a DNA methyltransferase inhibitor, which can lead to changes in gene expression and cellular processes.

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5-Aza (217 citations) 5-azacytidine (199 citations) 5-Azacytidine (5-Aza) (31 citations) Azacytidine (17 citations) Azacytidine (AZA) (3 citations)

10 protocols using 5 azacytidine aza

Engineered Anti-Mesothelin and Anti-CD25 Immunotoxins

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Clinical-grade anti-mesothelin SS1P and anti-CD25 LMB-2 [anti-Tac(Fv)-PE38] were manufactured and provided by Advanced BioScience Laboratories, Inc. (Kensington, MD). huSS1(Fab)-LR-GGS-LO10-PE24 (RG7787) was supplied by Roche Innovation Center Penzberg, Germany under a Cooperative Research and Development Agreement (#2791). Anti-mesothelin SS1(dsFv)-LR-GGS-PE24 (SS1-LR-GGS) and the anti-CD71 immunotoxin HB21(Fv)-PE40 were produced in our laboratory, according to a standard protocol [23 (link)]. Both SS1-LR-GGS and RG7787 are re-engineered low-immunogenic versions of SS1P that consist of a PE24 fragment. Specific modifications include removing the bulk of PE domain II, leaving a furin cleavage site, and adding a Gly–Gly–Ser (GGS)-based peptide linker after the furin cleavage site. RG7787 is further optimized for clinical use by replacing the mouse anti-mesothelin Fv (SS1) with a humanized Fab (huSS1), to increase size and therefore circulatory half-life, and by introducing seven mutations (R505A, R427A, R490A, R467A, D463A, R456A, and R538A) in the catalytic domain III of PE to silence B-cell epitopes [19 (link)]. 5-azacytidine (AZA) (Sigma) is a DNA methyltransferase inhibitor, and was dissolved in RPMI-1640 medium.

Hollevoet K., Mason-Osann E., Müller F, & Pastan I. (2015). Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line. PLoS ONE, 10(3), e0122462.

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Anticancer Drug Formulation and Cytotoxicity Assay

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A clinical formulation of cDDP was obtained from the UC San Diego Moores Cancer Center pharmacy. Cupric sulfate was obtained from Sigma (St. Louis, Mo.). The drugs were diluted to the desired concentrations in RPMI medium (Thermo Scientific, Logan, UT). The Detergent Compatible Protein kit was purchased from BioRad (Hercules, CA) and the tetrazolium compound WST-8 (Cell Counting Kit-8, CCK-8) from Dojindo Molecular Technologies (Rockville, MD). The demethylating agent 5-azacytidine (AZA) was purchased from Sigma. Mithramycin was purchased from Sigma.

Lin X., Shang X., Manorek G., Fofana M, & Stephen B. H. (2014). Integrin αV modulates the cellular pharmacology of copper and cisplatin by regulating expression of the influx transporter CTR1. Oncoscience, 1(3), 185-195.

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Epigenetic Modulation in Mouse Transplant

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Twelve- to 14-week-old B10.D2 (H-2d, Jackson Laboratories, Bar Harbor, USA) and BALB/cJ (H-2d, Jackson Laboratories) mice were used as donors and recipients, respectively. All mice were maintained in top-filtered cages in a standard animal facility and provided with sterilized food. Sterilized water, supplemented with 1 % Baytril (Bayer HealthCare, Diegem, Belgium), was given from day 0 to the end of the experiment. Water was changed every 2–3 days. All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège (Belgium), reference 1438. The “Guide for the Care and Use of Laboratory Animals”, prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, was followed carefully as well as European and local legislation. 5-Azacytidine (AZA, Sigma-Aldrich, St. Louis, MO) or Decitabine (DAC, Dacogen, Janssen-Cilag, Zug, Switzerland), solubilized in PBS (Lonza, Verviers, Belgium), were injected s.c. every 48 h from D + 10 to D + 30 at the dose of 0.5 mg/kg or 2 mg/kg for AZA and 0.75 mg/kg for DAC. Control mice did not receive any injection.

Fransolet G., Ehx G., Somja J., Delens L., Hannon M., Muller J., Dubois S., Drion P., Caers J., Humblet-Baron S., Delvenne P., Beguin Y., Conteduca G, & Baron F. (2016). Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease. Journal of Hematology & Oncology, 9, 53.

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Epigenetic Modulation of GABAT Activity

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For each cell line, 2 × 10⁵ cells were seeded in 12 wells and transduced with lentivirus, as previously mentioned. Additionally, they were treated with 5-Aza-2’-deoxycytidine (DAC, Sigma–Aldrich; Merck KGaA, Darmstadt, Germany) and 5-Azacytidine (AZA, Sigma–Aldrich) at concentrations of 500 nM and 1000, 1000, and 1200 nM based on IC50 of each cell line, respectively. For combined drug treatment, a medium concentration of rapamycin was used (RAPA, Sigma–Aldrich). The two cell lines were thereafter treated for 72 h by incubation with DAC, AZA, or combined RAPA with DAC or AZA. The GABAT enzyme activity was measured using the GABAT assay kit (Biomedical Research Service Center, University at Buffalo, Buffalo, NY, USA) following the protocol.

Zhao G., Li S., Wang Q., Wu W., Fu X., Zhu C., Wang W, & Wang X. (2022). ABAT gene expression associated with the sensitivity of hypomethylating agents in myelodysplastic syndrome through CXCR4/mTOR signaling. Cell Death Discovery, 8, 398.

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Modulatory Effects on Hematological Cell Lines

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HL-cell lines HDLM-2, KM-H2, L-428, L-540, L-1236, SUP-HD1, U-HO1 and AML cell line THP-1 are held by the DSMZ (Braunschweig, Germany) and cultivated as described [70 ]. Cell stimulations were performed for 16 h by treatment with 20 ng/ml recombinant human proteins IGF1, IFNG, IL2, IL6, IL7, IL12, IL21, IL22 or IL23 (R&D Systems, Wiesbaden, Germany), with inhibitory antibodies directed against IGF1, IFNGR1, IFNGR2, IL7, IL22 (R&D Systems), with 10 or 100 nM 5-Azacytidine (AZA, Sigma, Taufkirchen, Germany), 5 μM DZNep (Sigma), 10 μg/ml Trichostatin A (TSA, Sigma), 50 μM Resveratrol (Sigma), 0.5 μM ICBP112 (Sigma), 1 μM Epinephrine (Sigma), 1 μM Propranolol (Sigma), Doxorubicine (Sigma), AG490 (Sigma), and Etoposide (Sigma). Gene specific siRNA oligonucleotides and AllStars negative Control siRNA (siControl) were obtained from Qiagen (Hilden, Germany). Expression constructs for HLX, MSX1, STAT3, and GFP-tagged STAT3 were obtained from Origene (Wiesbaden, Germany). SiRNAs (80 pmol) and expression constructs/vector controls (2 μg) were transfected into 1 × 10⁶ cells by electroporation using the EPI-2500 impulse generator (Fischer, Heidelberg, Germany) at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation.

Nagel S., Pommerenke C., Meyer C., Kaufmann M., MacLeod R.A, & Drexler H.G. (2018). Aberrant expression of NKL homeobox gene HLX in Hodgkin lymphoma. Oncotarget, 9(18), 14338-14353.

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Establishing AZA-Resistant AML Cell Lines

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Two cell lines were used in this study. The MOLM-13 cell line (ACC 554) and the SKM-1 cell line (ACC 547) were derived from the peripheral blood of a 20-year-old and a 76-year-old man with AML following MDS, respectively (both supplied by Leibniz-Institute DSMZ-Deutsche Samsung von Microorganism und Zellkulturen GmbH, Braunschweig, Germany). The sensitive MOLM-13 and SKM-1 cell lines were adapted to 5-azacytidine (AZA, Sigma Aldrich, St. Louis, MO, USA) over a 6-month period with repeated passaging in medium containing AZA in stepwise increasing concentrations beginning at 0.1 nM up to final concentration of 1 μM. This procedure yielded AZA-resistant SKM-1/AZA and MOLM-13/AZA* cell variants. The MOLM-13/AZA cell variant was described previously [12 (link)]. The cell lines were cultured in RPMI 1640 medium with L-glutamine containing 12% fetal bovine serum (both from Gibco, Langley, OK, USA), 100,000 units/L penicillin, and 50 mg/L streptomycin (both from Sigma Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO₂.

Šimoničová K., Janotka L., Kavcova H., Sulova Z., Messingerova L, & Breier A. (2023). Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol. Cancers, 15(11), 3063.

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Evaluating Methylation and Expression

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To evaluate methylation and expression changes, 5′-azacytidine (aza) (Sigma-Aldrich, MO, USA) was used to treat the cells. For treatment with aza, SiHa and C33a cells were seeded at 3 × 10⁵ cells/mL in growth media. After overnight incubation, cells were further incubated with fresh media containing aza at the indicated final concentration (0–40 μM) for an additional 5 days, being replaced every 24 h until analysis.

Chalertpet K., Pakdeechaidan W., Patel V., Mutirangura A, & Yanatatsaneejit P. (2015). Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation. Cancer Science, 106(10), 1333-1340.

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Acetyl-CoA Carboxylase Pathway Regulation

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Antibodies for acetyl-CoA carboxylase 1 (ACC1), phospho-ACC1 (S79), AMPK, phospho-AMPK (T172), Sirt1, and Sirt3 (C73E3) were obtained from Cell Signaling Technologies (Beverly, MA, USA), antibodies against CD38 and FOXO1 were from Abcam (Cambridge, MA, USA), antibodies for SLC22A3 and β-Actin were purchased from Sigma-Aldrich (St. Louis, MO, USA) and antibody for Ac-FOXO1 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Secondary antibodies for goat-anti-mouse and goat-anti-rabbit were purchased from BioRad (Hercules, CA, USA). The NAMPT inhibitor FK866 was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Nicotinamide adenine dinucleotide (NAD⁺), nicotinamide mononucleotide (NMN), doxycycline hyclate, and puromycin dihydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). ¹⁴C-Acetate was purchased from PerkinElmer (Boston, MA, USA) and ¹⁴C – Choline from American Radiolabeled Chemicals (St. Louis, MO, USA). Retinoic acid (all-trans-Retinoic acid; ATRA) and 5-Azacytidine (Aza) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All primers for qPCR were purchased from Integrated DNA Technologies (San Jose, CA, USA).

Chmielewski J.P., Bowlby S.C., Wheeler F.B., Shi L., Sui G., Davis A.L., Howard T.D., D’Agostino RB J.r., Miller L.D., Sirintrapun S.J., Cramer S.D, & Kridel S.J. (2018). CD38 Inhibits Prostate Cancer Metabolism and Proliferation by Reducing Cellular NAD+ Pools. Molecular cancer research : MCR, 16(11), 1687-1700.

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Epigenetic Modulation and p53 Activation

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Cells were cultured in T75 dishes (Greiner, 658170) and treated with 500 nM 5-azacytidine (AZA, Sigma-Aldrich) or PBS. Media and AZA were replaced each day for five days (Fig. 1B). The cells were then split and allowed to reattach. ITF-2357 was added at a concentration of 100 nM and treatment was continued for two days. The sequential treatment of these cells by these drugs was optimized by Topper et al. (28 (link)). The A2780 p53 ChIP-seq samples were treated with 500 nM AZA (Sigma-Aldrich) or PBS for three days and 10 uM Nutlin-3A (Cayman Chemical 18585) or DMSO for 6 hours.

McDonald J.I., Diab N., Arthofer E., Hadley M., Kanholm T., Rentia U., Gomez S., Yu A., Grundy E.E., Cox O., Topper M.J., Xing X., Strissel P.L., Strick R., Wang T., Baylin S.B, & Chiappinelli K.B. (2021). Epigenetic therapies in ovarian cancer alter repetitive element expression in a TP53-dependent manner. Cancer research, 81(20), 5176-5189.

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Epigenetic Modulation via Decitabine and 5-azacytidine

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Human cell lines were cultured as described in Supplemental Experimental Procedures. Decitabine (DAC, Sigma-Aldrich) was prepared at 10 mM in DMSO. 5-azacytidine (AZA, Sigma-Aldrich) was prepared at 500 µM in PBS. PARPis Veliparib (ABT888; 200 mM in water) was obtained from Enzo Life Sciences, and BMN 673 (5 mM in DMSO) was obtained from Abmole BioScience, (Kowloon, Hong Kong).

Muvarak N.E., Chowdhury K., Xia L., Robert C., Choi E.Y., Cai Y., Bellani M., Zou Y., Singh Z.N., Duong V.H., Rutherford T., Nagaria P., Bentzen S.M., Seidman M.M., Baer M.R., Lapidus R.G., Baylin S.B, & Rassool F.V. (2016). Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents – A Potential Therapy for Cancer. Cancer cell, 30(4), 637-650.

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