5 aza 2 deoxycytidine dac

MDA-MB-231 and MCF-7 breast cancer cell lines were provided by the cell resource center of the Chinese Academy of Medical Sciences (Beijing, China). MDA-MB-231 cells were grown in DMEM/F12 (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA). MCF-7 breast cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Gibco, Rockville, MD, USA). Cells were cultured at 37 °C in 5% CO₂ to 80%–90% confluence and were subjected to DMEM/F12 or DMEM supplemented with 2% FBS, containing 0.01, 0.02, 0.1, 0.5 or 1 mM lidocaine (Sigma–Aldrich, St. Louis, MO, USA), 10 μM 5-aza-2'-deoxycytidine (DAC) (Sigma–Aldrich) or (and) 0.2 μM cisplatin (Sigma–Aldrich for various hours for the DNA methylation sequencing, cell viability or cell apoptosis assay, and cell colony forming assay. DAC or cisplatin was utilized to as a positive demethylation agent or as an apoptosis inducer. To abrogate the expression of RARβ2 or RASSF1A, siRNA–RARβ2 (5'-CAGC UGAG UUGG ACGA UCU-3'), siRNA–RASSF1A (5'-GAC CUC UGU GGC GAC UUCA-3') or siRNA control (5'-AGCG AATT AGCT TGCC GTG-3') was synthesized by GenePharma Technology (Shanghai, China) and was transfected by lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) with a concentration of 50 nM according to the manufacurer’s guidance.

NPC cell lines were treated with or without 10 μmol/L 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich, Munich, Germany) for 72 h, with the drug/media replaced every 24 h. DNA was isolated using the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany), then 1–2 μg DNA was treated with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s instructions. Bisulfite pyrosequencing primers were designed using PyroMark Assay Design Software 2.0 (Qiagen), and were: PCR forward primer: 5′-GAATTTAGGTTAGGGATAGTTTGAT-3′ (F); PCR reverse primer: 5′-CCAAAAAAAAAATATTTCAATACC-3′ (R); sequencing primer: 5′-GG ATAGTTTGA TAGAAATAAAATG-3′(S). The PyroMark Q96 System (Qiagen) was used for the sequencing reactions and to quantify methylation.

Human hepatoma HepG2, HuH7, and JHH1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum at 37 °C in a humidified atmosphere containing 5 % CO₂. In order to reverse DNA methylation, the cells were treated with 0.5 or 5 μM 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. After DAC treatment, the cells were treated with trichostatin A (TSA; Sigma-Aldrich) at 200 nM (HepG2 and HuH7 cells) or 20 nM (JHH1 cells) for 24 h.

NPC cell lines (6 × 10⁵cells) were seeded on 100 mm culture dishes. After culturing for 24 h, the cells were treated with or without 10 μmol/L 5-aza-2′-deoxycytidine (DAC, Sigma Aldrich, Munich, Germany) for 72 h with replacing the drug and medium every 24 h. Subsequently, DNA was extracted using the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For bisulfite pyrosequencing analysis, 1–2 μg DNA was treated by sodium bisulfite using the EpiTect Bisulfite Kit (Qiagen). The PyroMark Assay Design Software 2.0 (Qiagen) was employed to design bisulfite pyrosequencing primers for ARNTL as follow: PCR forward: 5′-GGAAGGGGAGTGTTGGATAT-3′; PCR reverse: 5’-CCAAAACAACCCTAAATAACC-3′; sequencing primer: 5′- GGATATAGGAGTTTGTTGTTAA-3’.The PyroMark Q96 System (Qiagen) was adopted to conduct sequencing reaction and quantify methylation level.

The hADSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China). Briefly, the hADSCs were isolated and purified from fresh human adipose tissues donated from healthy adults less than 45 years of age after liposuction. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, United States) containing 10% fetal bovine serum and cultured at 37°C in a humidified atmosphere of 5% CO₂. Osteogenic induction medium was purchased from SALILA (SALILA, Guangzhou, China). The medium was changed every 2 days. To inhibit DNA methylation, hADSCs were treated with 5 μmol/L 5-aza-2′-deoxycytidine (DAC, Sigma Aldrich, Munich, Germany). To inhibit NOTCH signaling, cells were treated with 2 μM DAPT (Selleck). Protein deacetylation was inhibited using 20 μM OSS_128167 (Selleck). For the cycloheximide (CHX)-chase assay, cells were treated with 100 g/mL CHX (Sigma-Aldrich, St. Louis, MO, United States) for the indicated hours in the absence or presence of 5 g/mL actinomycin D (Sigma-Aldrich, St. Louis, MO, United States) and western blot analysis was performed.