5-Aza-2'-deoxycytidine (5-Aza-CdR) was obtained from Sigma. Cells were seeded 24 hours before treatment and drugs were administered as noted in the figure legends. Predesigned siRNAs specific for LPHN2 were purchased from M-biotech (Hanam, Korea) and control siRNAs with scrambled sequences were obtained from Qiagen. To confirm whether LPHN2 expression affects chemosensitivity, siLPHN2 was transfected into DKO, SNU484, and SW480 cells for 8 hours using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were subsequently plated into 96-well plates and cell viability assays were performed in the presence of cisplatin.
5 aza 2 deoxycytidine 5 aza cdr
Manufactured by Merck Group
Sourced in United States, Germany
5-aza-2′-deoxycytidine (5-Aza-CdR) is a synthetic nucleoside analog. It is used as a research tool in molecular biology and biochemistry laboratories.
Automatically generated - may contain errors
Lab products found in correlation
5 aza cdr, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pcr mycoplasma detection set, by Takara Bio (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Trichostatin a tsa, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Trichostatin a, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (1 mentions)
Keratinocyte sfm, by Thermo Fisher Scientific (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
S adenosylmethionine sam, by New England Biolabs (1 mentions)
H2o2 treatment, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Smartpool ntsr1 sirna, by Horizon Discovery (1 mentions)
Sr48692, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Km12c, by American Type Culture Collection (1 mentions)
3 deazaneplanocin a dznep, by Merck Group (1 mentions)
31 protocols using 5 aza 2 deoxycytidine 5 aza cdr
1
Sensitizing Cells to Cisplatin
2
Cell Line Characterization and Demethylation
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Prostate cell lines, LNCaP, 22RV1, DU145, PC-3 (malignant), and RWPE (benign) were used for in vitro studies. LNCaP and 22Rv1 cells were grown in RPMI 1640, whereas DU145 and PC-3 cells were maintained in MEM and 50% RPMI-50% F-12 medium, while RWPE was cultured in Keratinocyte-SFM, containing human recombinant Epidermal Growth Factor 1-53 and Bovine Pituitary Extract (GIBCO, Invitrogen, Carlsbad, CA, USA), respectively. HEK293Ta were maintained in DMEM. All basal culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (GIBCO, Invitrogen, Carlsbad, CA, USA). Cells were maintained in an incubator at 37 °C with 5% CO2. All cell lines were G-banding karyotyped (for validation) and routinely tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set, Clontech Laboratories).
One micromolar of the DNA methyltransferases inhibitor 5-aza-2-deoxycytidine (5-Aza-CdR; Sigma-Aldrich, Schnelldorf, Germany) was used for DNA demethylation. Cells were harvested and RNA extracted after 72-h exposure to the demethylating agent.
One micromolar of the DNA methyltransferases inhibitor 5-aza-2-deoxycytidine (5-Aza-CdR; Sigma-Aldrich, Schnelldorf, Germany) was used for DNA demethylation. Cells were harvested and RNA extracted after 72-h exposure to the demethylating agent.
3
Optimizing Epigenetic Modulation in Lung Cancer
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Cultivated lung cancer cells were treated either with 5-Aza-2′-deoxycytidine (5-Aza-CdR) (Sigma) at different concentrations dissolved in culture medium. methylation specific PCR (MSP) and MTT assays were used to select the 5-Aza-CdR concentration which enables β-catenin promoter CpG island demethylation without significant effects on cell growth (7 µmol/L) which was then used for subsequent demethylation processing and cells were collected after continued culture for 48 h.
4
Epigenetic and Cytotoxicity Modulation
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5-Aza-2′deoxycytidine (5-aza-CdR; Sigma-Aldrich) and S-adenosylmethionine (SAM; NEB, Ipswich, MA) was added to cells in fresh medium daily at indicated concentrations for 3 or 5 days or equal volume DMSO or 0.005 M H2SO4 plus 10% ETOH respectively. JQ1 (250–500 nM; Bradner Lab; Dana-Farber Cancer Institute) or DMSO was added to cells for 16 hours. Gemcitabine (Sigma-Aldrich) or 3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2 H)-isoquinolinone (DPQ; Sigma-Aldrich) treatment or equal volume vehicle control was added once for 72 hours.
For H2O2 treatment (Sigma-Aldrich), cells were plated in a 96-well plate at 2000 cells/well. Medium was changed each day to normal growth 500 μM H2O2 containing medium. Cell density was measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Fisher Scientific) according to manufacturer’s protocol.
For H2O2 treatment (Sigma-Aldrich), cells were plated in a 96-well plate at 2000 cells/well. Medium was changed each day to normal growth 500 μM H2O2 containing medium. Cell density was measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Fisher Scientific) according to manufacturer’s protocol.
5
ATRA and 5-Aza-CdR Induction of RARβ2 Expression
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All-trans-retinoic acid (Sigma-Aldrich, Dorset, UK), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10−2 M, and stored in dark at −70 °C, was diluted in growth medium to a final concentration of 10−6 M immediately before each experiment. Control cultures received the same amount of DMSO as treated cultures. 5-aza-2′-deoxycytidine (5-Aza-CdR) (Sigma, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 2 × 10−3 M, stored in small aliquots the dark at −70 °C. Cell lines were treated with 0.5 µM of 5-Aza-CdR with media refreshed every 24 h for 5 consecutive days before genomic DNA and total RNA were extracted and tested for methylation status as well as restoration of RARβ expression. RARβ2 induction was analysed in the cells treated using 1, 5, or 10 µM of ATRA for 3 days. Untreated cells were used as an experimental control, and cells treated with a working concentration of DMSO were used as solvent control.
6
Cell Culture Conditions for Endometrial Cell Lines
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EC cell lines HEC-1-B (ATCC® HTB-113), HEC-1-A (ATCC® HTB-112), and KLE (ATCC® CRL-1622) were purchased from American Type Culture Collection (ATCC Manassas, VA, USA). HEC-1-B cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with non-essential amino acids (NEAAs), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. HEC-1-A cells were grown in McCoy’s 5a (Modified) Medium with 10% FBS. KLE cells were cultured in DMEM/F12 (Nutrient mixture F12) supplemented with 10% FBS and 1% penicillin-streptomycin. Additionally, the immortalized endometrial stromal cell line hEM15A cells (Institute of basic medical sciences CAMS, Beijing, China) were cultured in the mixture of DMEM/F12 Ham’s F12/DME Medium (DME H-16/F-12 50% Mixture w/o HEPES, with NEAA Medium) (1: 1) encompassing 15% FBS. All cells were cultured in an incubator at 37°C with 5% CO2. 5-aza-2′-deoxycytidine (5-Aza-CdR; Sigma-Aldrich Chemical Company, St Louis, MO, USA) was diluted in dimethylsulfoxide and used at a concentration of 2 μM.
7
Epigenetic Modulation of Cervical Cancer Cells
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1.0 × 105/well SiHa, HeLa and C33A cells were cultured in 6-well plates in DMEM with 10% FBS, after 24 h, the medium was replaced with fresh medium containing 0 μM, 2.5 μM or 5 μM 5-Aza-2′-deoxycytidine (5-Aza-CdR) (Sigma, USA). The medium containing 5-Aza-CdR was replaced every 24 h during a 72-h period [15 (link)].
8
Epigenetic Modulation of MUC Gene Expression
9
CRC Cell Line Characterization and Treatment
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The human CRC cell lines KM12c, Caco2, DLD1, HT29, HCT116, and SW480 were used in these studies. Cell lines were authenticated in February and May 2016 at Genetica DNA Laboratories (Cincinnati, OH). KM12c cells were kindly provided by Dr Isaiah J. Fidler (M.D. Anderson Cancer Center, Houston, TX, USA); other cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). KM12c cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% non-essential amino acids and 2% MEM essential vitamins. Caco2 cells were incubated in MEM supplemented with 15% FBS, 1% sodium pyruvate and 1% non-essential amino acids. DLD1 cells were grown in RPMI-1640 with 10% FBS. HT29 and HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% FBS. SW480 cells were cultured in DMEM with 10% FBS. Cells were maintained at 37°C in a humidified 5% CO2 incubator. The DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-CdR), and a selective inhibitor for NTSR1, SR48692, were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Transfections with non-targeting control and SMARTPool NTSR1 siRNA (Dharmacon, Lafayette, CO, USA) were performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) as previously described (16 (link)).
10
Epigenetic Regulation of MEG3 Expression
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The DNA demethylating agent 5-aza-2-deoxy-cytidine (5-aza-CdR) was obtained from Sigma-Aldrich (MO, USA) and diluted in dimethyl sulfoxide (DMSO, MP Biomedicals, California, USA). Total 1 × 105 cells were seeded in six-well culture plate and treated with 0, 5, 10 μmol/L 5-aza-CdR for next 5 days. The medium was replaced with the same concentration of 5-aza-CdR every day. Then cells were collected and prepared for qRT-PCR and MSP to measure the expression of MEG3 and the methylation status of the MEG3 promoter respectively. For CCK-8 assay, we treated cells with 10 μmol/L 5-aza-CdR for 5 days, following transfection of si-MEG3 and compared proliferation ability with cells which were treated with 5-aza-CdR only.
3-Deazaneplanocin A (DZNep), an inhibitor of the histone methyltransferase EZH2, was purchased from Sigma-Aldrich (MO, USA). The cells were seeded at 1 × 105 cells per well and treated with DZNep at 0, 1 and 5 μmol/L for 5 days. After that, cells were harvested for qRT-PCR and Western Blot.
3-Deazaneplanocin A (DZNep), an inhibitor of the histone methyltransferase EZH2, was purchased from Sigma-Aldrich (MO, USA). The cells were seeded at 1 × 105 cells per well and treated with DZNep at 0, 1 and 5 μmol/L for 5 days. After that, cells were harvested for qRT-PCR and Western Blot.
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