5 azacytidine 5 aza

Cell lines cultured in DMEM with 10% FBS, 24 hours later, the medium was replaced with fresh medium containing 1, 5, or 10 mM 5-Azacytidine (5-Aza; Sigma-Aldrich, Inc, St Louis, MO) or an equal volume of vehicle (PBS). The medium containing drug or vehicle was replaced every 24 hours during a 72-hour period.

RRx-001 was obtained from Orbital ATK (Dulles, VA, USA). The DNA methyltransferase inhibitor 5-azacytidine (5-AZA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ruxolitinib was purchased from Selleck Chemical (Houston, TX, USA). RRx-001, 5-AZA and Ruxolitinib solutions were freshly prepared on the experimental day by dissolving in DMSO and then diluting with cell culture medium. The final concentration of DMSO was 0.025%.

All cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and grown according to the ATCC instructions. Total RNA from human prostate tissues was obtained from Clontech (Mountain View, CA, USA). 5-Azacytidine (5-Aza, Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in PBS before use. A vehicle control consisting of culture medium alone was included in the analysis. MCF7 and PC3 cells were pre-cultured for 24 h, then treated with 1 µM 5-Aza for 72 h. Cells were collected by centrifugation, then genomic DNA and RNA were extracted and analyzed.

EOC cell lines A2780, CAOV3, C13*, ES2, HO8910, OV2008 and SKOV3 were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). All cells were cultured at 37 °C with DMEM/F12 medium (Gibco, Grand Island, NY, USA) containing 10% fetal calf serum (Gibco, USA) under 5% CO2 atmosphere. The demethylation agent 5-Azacytidine (5Aza, Sigma-Aldrich Corp, St. Louis, MO, USA) and the inhibitor of H3K27me3-GSK126 (Cayman Chemical, Ann Arbor, Michigan, USA) were added to fresh culture medium of ovarian cancer cells with final concentrations of 10 uM and 500 nM, respectively. Cells were collected and analyzed by subsequent experiments after 72 h of culture.

Chromatin was isolated as previously described [27 ]. For de-methylation studies, cells were treated with 5 μM 5′-aza-cytidine (5′-aza; Sigma Aldrich; cat. # A2385-100MG) every 17 h for 5 consecutive days. Fifteen micrograms of chromatin was precipitated with 8 μg mouse anti-Kaiso 6F monoclonal [28 ], 4 μg mouse non-specific IgG (Abcam cat. #ab37415) or 4 μg rabbit anti-Histone H3 (Abcam cat. #ab1791) antibodies. Precipitated DNA fragments were resuspended with nuclease-free water and PCR amplified. Primers used for ChIP are listed in Table 1.

5-Azacytidine (5-AZA, Sigma Aldrich, St Louis, MO, USA) was used to assess the effect of demethylation in cell lines. Cells were plated with a concentration of 25,000 cells/cm² (12,500 cells/mL for JHH6) for 24 h then exposed to 5-AZA with concentrations ranging from 2 uM to 5 mM for another 24 h. DMSO concentration was calculated to be 0.3% in each treatment. Cell viability was determined by 3(4,5-dimethyl thiazolyl-2)-2,5 diphenyltetrazolium assay (MTT, Sigma Aldrich). Total protein and RNA extracts were collected for proteomic and gene analysis, respectively.

The DNA methyltransferase inhibitor 5-Azacytidine (5-aza) was purchased from Sigma-Aldrich and was reconstituted at 10 mM using dimethyl sulfoxide (DMSO; Sigma-Aldrich). For 5-aza pretreatment, the medium was changed to a freshly made culture medium containing 10 µM 5-aza. After 24 h, the 5-aza containing culture medium was refreshed for additional 24 h (total treatment 48 h). DMSO alone was used as control at 0.1% (v/v) concentration. Cells pretreated with 5-aza or DMSO were cultured in standard medium for 72 h and then subjected to analyses, unless otherwise specified. When indicated, 5-aza pretreatment was performed in the presence of the mTOR inhibitor rapamycin (0.5 mM; Sigma-Aldrich) or of human recombinant Wnt3a (100 ng/ml; R&D Systems, Minneapolis, MN, United States).