5 aza 2 deoxycytidine aza

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom

5-aza-2'-deoxycytidine (AZA) is a chemical compound used in laboratory research. It is a synthetic analog of the DNA nucleoside cytidine. AZA is commonly used as a DNA methylation inhibitor in cell culture experiments.

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47 protocols using 5 aza 2 deoxycytidine aza

Epigenetic Modulation of hiPSCs

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The Alphoid^tetO-HAC-GFP hiPSCs were cultured for 24 h in mTeSR-1 media in the presence of DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine (AZA) (Sigma-Aldrich, St. Louis, MO, USA) or histone deacetylase inhibitor trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 5–10 μM and 0.5 μM, respectively. The Alphoid^tetO-HAC-GFP hiPSCs were cultured for 72 h in the presence of 100–400 nM AZA or 19–38 nM TSA. The GFP-expression in the living cells was monitored by fluorescent and phase contrast light microscopy (EVOS FL Auto Imaging System, Thermo Fisher Scientific, Waltham, MA, USA).
The Alphoid^tetO-HAC-GFP hiPSCs were cultured for 24 h in mTeSR-1 media in the presence of DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine (AZA) (Sigma-Aldrich, St. Louis, MO, USA) or histone deacetylase inhibitor trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 5–10 μM and 0.5 μM, respectively. The Alphoid^tetO-HAC-GFP hiPSCs were cultured for 72 h in the presence of 100–400 nM AZA or 19–38 nM TSA. The GFP-expression in the living cells was monitored by fluorescent and phase contrast light microscopy (EVOS FL Auto Imaging System, Thermo Fisher Scientific, Waltham, MA, USA).

Sinenko S.A., Skvortsova E.V., Liskovykh M.A., Ponomartsev S.V., Kuzmin A.A., Khudiakov A.A., Malashicheva A.B., Alenina N., Larionov V., Kouprina N, & Tomilin A.N. (2018). Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications. Cells, 7(12), 261.

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Transcriptional Profiling of Melanoma Cell Lines

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Total RNA samples of paired melanoma cell lines IGR39 and IGR37 were extracted and sent to the IRB Functional Genomic Core External Service (Barcelona). Once samples had been reversed-transcribed and labeled, they were hybridized onto Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Hybridization, washing, staining and scanning were performed using Affymetrix GeneChip system instruments and protocols. For breast and colon cancer cell lines, expression data were obtained from the Gene Expression Omnibus (GSE11683, GSE10843 and GSE57083). For qRT–PCR experiments, total RNA was extracted using Trizol reagent and retrotranscribed using the ThermoScript RT-PCR system (Invitrogen). The reaction was carried out following the methods for use of SYBR Green (Applied Biosystems), and B2M, GAPDH and ACTB were used as housekeeping genes to enable normalization. For primer sequences please refer to Supplementary Table 3. We performed reactivation treatments with the demethylating agent 5-aza-2′-deoxycytidine (AZA; Sigma) at 0.5 μM for 72 h. For immunoblotting assays, we extracted total protein using RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA and EGTA, 1% NP40, 0.5% of sodium deoxycholate, 0.1% of SDS, and protease and phosphatase inhibitors from Roche). All the antibodies used in this study are described in Supplementary Table 3.

Vizoso M., Ferreira H.J., Lopez-Serra P., Javier Carmona F., Martínez-Cardús A., Girotti M.R., Villanueva A., Guil S., Moutinho C., Liz J., Portela A., Heyn H., Moran S., Vidal A., Martinez-Iniesta M., Manzano J.L., Fernandez-Figueras M.T., Elez E., Muñoz-Couselo E., Botella-Estrada R., Berrocal A., Pontén F., van den Oord J., Gallagher W.M., Frederick D.T., Flaherty K.T., McDermott U., Lorigan P., Marais R, & Esteller M. (2015). Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR. Nature medicine, 21(7), 741-750.

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Synergistic Gene Reactivation by DNMT and HDAC Inhibitors

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5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase (DNMT) inhibitor, makes DNMT inactivation through DNMT covalent bonding with thiol on cysteine residues, resulting in reactivation genes silenced by promoter methylation. Trichostatin A (TSA), a histone deacetylase inhibitor, plays a significant role in controlling the tightness of DNA around histone. Combination treatment of TSA and 5-Aza-dC results in the synergistic activation of methylated genes.
As previously described [32 (link)], breast cancer cell lines were treated with 10 mmol/L 5-aza-2-deoxycytidine (Aza) (Sigma-Aldrich, Steinheim, Germany) for 3 days and further treated with or without 100 nmol/L TSA (Sigma-Aldrich) for an additional 24 h.

Mu J., Hui T., Shao B., Li L., Du Z., Lu L., Ye L., Li S., Li Q., Xiao Q., Qiu Z., Zhang Y., Fan J., Ren G., Tao Q, & Xiang T. (2017). Dickkopf-related protein 2 induces G0/G1 arrest and apoptosis through suppressing Wnt/β-catenin signaling and is frequently methylated in breast cancer. Oncotarget, 8(24), 39443-39459.

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Epigenetic Regulation of Cell Proliferation

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The reagents used were as follows: Palbociclib (0.1 μM or 0.3 μM, Selleck Chemicals, Cat #S1116), 4-Hydroxytamoxifen (0.5 μM, Sigma, Cat #h7904), Trichostatin A (0.3 μM, Sigma, Cat #T8552), Entinostat (1 μM, Cayman Chemical, Cat #13284), DZNep (1 μM, Cayman Chemical, Cat #13828), 2-PCPA (200 μM, Cayman Chemical, Cat #10010494), GSK-LSD1 (1 μM, Santa Cruz, Cat #sc-490345), and 5-Aza-2′-deoxycytidine (AZA) (10 μM, Sigma, Cat #A3656). VT02956 and VT02484 were provided by Vivace Therapeutics.

Ma S., Tang T., Probst G., Konradi A., Jin C., Li F., Gutkind J.S., Fu X.D, & Guan K.L. (2022). Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER+ breast cancer. Nature Communications, 13, 1061.

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Generation of Induced Decidual Natural Killer Cells

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Conversion of pNK cells to idNK cells were done as previously described [36 ]. Briefly, NK RosetteSep enriched pNK cells were seeded at 1 X 10⁶ cells/ml in IL-15 complete media with the addition of recombinant human TGF-β1 (PeproTech) at 2 ng/ml, 1 μM 5-aza-2-deoxycytidine (Aza) (Sigma-Aldrich, St Louis, MO), and maintained in culture 7 days (for injection at gd9) or 10 days (for injection at gd12) at 37°C in humidified incubators in a 1% O₂ and 5% CO₂ atmosphere. To assess conversion efficiency, the percentage of CD9⁺ KIR⁺ cells among the CD16^- CD56^Bright and CD16⁺ CD56^Dim idNK populations was evaluated by flow cytometry after 6 or 7 days in culture. For microarray gene expression profiling, CD3^- CD16^- CD56^Bright CD9⁺ KIR⁺ idNK and CD3^- CD16⁺ CD56^Dim CD9⁺ KIR⁺ idNK cells were further purified from the idNK cell bulk preparation by FACS.

Cavalli R.C., Cerdeira A.S., Pernicone E., Korkes H.A., Burke S.D., Rajakumar A., Thadhani R.I., Roberts D.J., Bhasin M., Karumanchi S.A, & Kopcow H.D. (2016). Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice. PLoS ONE, 11(10), e0164353.

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Regulation of Retinal Endothelial Cells

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Human retinal endothelial cells (Cell system, Kirkland, WA) from 7–8^th passage were incubated in NG or HG media containing 1% fetal calf serum, 9% Nu-serum and 1 µg/mL endothelial growth supplement for 4 days^{13 (link),14 (link)}. Cell incubated in high glucose were divided into two groups, cells in group 1 remained in high glucose for eight days, in the absence or presence of 1 µM 5-aza-2′-deoxycytidine (Aza; Sigma-Aldrich Corp, St. Louis, MO, USA), respectively. In group 2, after four days of high glucose exposure, the cells were incubated in normal glucose for four additional days, in the absence or presence of Aza (HG-NG and HG-NG/Aza). For osmotic/ metabolic control, 20 mM D-glucose was replaced by 20mM L-glucose (L-Gl). As reported previously, under these incubation conditions, morphology of HRECs was maintained^{22 (link)}.

Kowluru R.A, & Mohammad G. (2020). Epigenetics and Mitochondrial Stability in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy. Scientific Reports, 10, 6655.

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Reactivating FOXO1 Transcription in Cell Lines

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The cell lines HeLa, SiHa, ME180 and SW756 were treated with 5-Aza-2'-deoxycytidine (AZA) (Sigma, USA) and HDAC inhibitor Trichostatin A (TSA) (Sigma, USA) as described previously in 25 (link) to reactivate FOXO1 transcription. For cell proliferation, cell viability and apoptosis, cells were initially grown in 96-well plate (1x10⁴ cells/well) and 6-well plate (1x10⁵ cells/well) for 24hr, followed by treatment with 10µM and 25 µM of LY294002 for 48hr.

Prasad S.B., Yadav S.S., Das M., Govardhan H.B., Pandey L.K., Singh S., Pradhan S, & Narayan G. (2014). Down Regulation of FOXO1 Promotes Cell Proliferation in Cervical Cancer. Journal of Cancer, 5(8), 655-662.

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Breast Cancer Cell Line DNA Demethylation

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Breast cancer cell lines were subjected to DNA demethylation treatments, as previously described [8 (link)]. Cell lines were treated with 10 mM 5-aza-2′-deoxycytidine (Aza) (Sigma-Aldrich, Steinheim, Germany) for 3 days, and then treated with 100 nM trichostatin A (TSA) (Sigma-Aldrich, Steinheim, Germany) for an additional 24 h.

Yin X., Xiang T., Mu J., Mao H., Li L., Huang X., Li C., Feng Y., Luo X., Wei Y., Peng W., Ren G, & Tao Q. (2016). Protocadherin 17 functions as a tumor suppressor suppressing Wnt/β-catenin signaling and cell metastasis and is frequently methylated in breast cancer. Oncotarget, 7(32), 51720-51732.

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qRT-PCR and Immunoblotting Analysis

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For qRT–PCR experiments, total RNA was extracted using Trizol^® reagent and retrotranscribed using the ThermoScript™ RT–PCR System (Invitrogen). The reaction was carried out following the methods for use of SYBR Green (Applied Biosystems), and HPRT were used as housekeeping gene to enable normalization. Reactivation treatments with the demethylating agent 5-aza-2′-deoxycytidine (AZA; Sigma) were performed at 1 μM for 72 h. For immunoblotting assays, total protein was extracted using RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA and EGTA, 1% NP40, 0.5% of sodium deoxycholate, 0.1% of SDS, and protease and phosphatase inhibitors from Roche), and specific antibodies against target proteins are listed in the enclosed table.

Nogales V., Reinhold W.C., Varma S., Martinez-Cardus A., Moutinho C., Moran S., Heyn H., Sebio A., Barnadas A., Pommier Y, & Esteller M. (2015). Epigenetic inactivation of the putative DNA/RNA helicase SLFN11 in human cancer confers resistance to platinum drugs. Oncotarget, 7(3), 3084-3097.

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Quantitative RNA Analysis with AZA Treatment

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For real-time quantitative reverse transcription-PCR experiments, total RNA was extracted using the SimplyRNA kit (Promega) on a Maxwell RSC device (Promega) and retrotranscribed using the ThermoScript RT-PCR system (ThermoFisher) with oligo(dT) primers. The reaction was carried out following the methods for the use of SYBR Green (Applied Biosystems), and GAPDH was used as housekeeping genes to enable normalization. Primers for qPCR are listed in the supplementary Key Resources Table. Reactivation treatments with the demethylating agent 5-aza-2′-deoxycytidine (AZA; Sigma) were performed at 0.5 µM for 72 h. For immunoblotting assays, we extracted cell pellets and brain white matter samples with 300 µL of RIPA buffer containing protease and phosphatase inhibitors cocktail cOmplete™ (Roche). Antibodies used in this study are described in supplementary Key Resources Table.

Janin M., Ortiz-Barahona V., de Moura M.C., Martínez-Cardús A., Llinàs-Arias P., Soler M., Nachmani D., Pelletier J., Schumann U., Calleja-Cervantes M.E., Moran S., Guil S., Bueno-Costa A., Piñeyro D., Perez-Salvia M., Rosselló-Tortella M., Piqué L., Bech-Serra J.J., De La Torre C., Vidal A., Martínez-Iniesta M., Martín-Tejera J.F., Villanueva A., Arias A., Cuartas I., Aransay A.M., La Madrid A.M., Carcaboso A.M., Santa-Maria V., Mora J., Fernandez A.F., Fraga M.F., Aldecoa I., Pedrosa L., Graus F., Vidal N., Martínez-Soler F., Tortosa A., Carrato C., Balañá C., Boudreau M.W., Hergenrother P.J., Kötter P., Entian K.D., Hench J., Frank S., Mansouri S., Zadeh G., Dans P.D., Orozco M., Thomas G., Blanco S., Seoane J., Preiss T., Pandolfi P.P, & Esteller M. (2019). Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program. Acta Neuropathologica, 138(6), 1053-1074.

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