5 aza 2 deoxycytidine decitabine

5-aza-2′-deoxycytidine (decitabine) was purchased from Sigma. Cells were treated with decitabine for 48 hours at the concentrations indicated in the figures/figure legends.

5-Azacytidine (Sigma-Aldrich; A1287), 5-Aza-2’-Deoxycytidine/Decitabine (Sigma-Aldrich; A3656) and Pyrimidin-2-one Beta-Ribofuranoside/Zebularine (Sigma-Aldrich; Z4775) were freshly resuspended in DMSO at the concentration recommended by supplier. The suspension was further diluted in the culture media for the experiment to final working concentration of up to 10 µM. Cells were treated with media containing 5-Azacytidine (or derivatives) replaced every 24 h to ensure proper action of the drug(s) for long-term expositions. Proteasome inhibitor MG132 was purchased from Merck Millipore (InSolution^TM MG132) and it was used at final concentration of 10 µM for 4 h. Caffeine was purchased from Sigma-Aldrich (C0750) and it was used at final concentration of 1 mM for 24 h.
Control scrambled siRNAs and siRNAs directed against ZBTB38, USP9X, p53, CDKN1C and DNMT1 were purchased from Thermo Fisher Scientific and some were validated in previous studies^{31 (link),32 (link)}. siRNAs were delivered in adherent cells using Lipofectamine 3000 following protocols provided by the manufacturer (Thermo Fisher Scientific). Cells in suspension (K562, MOLM-14 and THP-1) were electroporated using the Neon^® transfection system (Thermo Fisher Scientific) using the kit MPK10096 and the following parameters: pulse voltage at 1450 V, pulse width 10 milliseconds and a total of 3 sequential pulses.

Affymetrix Human Genome U133A arrays were used to quantify gene expression by the Duke DNA Microarray Facility using total RNA isolated from 16 paired primary-recurrent frozen ovarian tumor samples. This data is publicly available through the Duke Digital Data Repository (22 (link)). An independent Affymetrix U133A gene expression dataset from tumors of women who lived <3 years (n= 26) or >7 years (n= 21) post-diagnosis was used for validation and derived from previously published data (GSE51820) (21 (link)). Gene expression for 26 OC cell lines treated for 72 h with 5 µM 5’-aza-2’-deoxycytidine (Decitabine; Sigma-Aldrich; St. Louis, MO; #A3656) was quantified using the Affymetrix HT Human Genome U133A Array and is available from the NCBI GEO web site, accession GSE25428. This data was analyzed using a paired student’s t-test.

For the inhibition assay 5-Aza-2’-cytidine (Sigma) was used in two different concentrations of 10μM and 3 μM whereas 5-Aza-2’deoxycytidine/Decitabine (Sigma) was used in three different concentrations of 1.5μM, 1 μM and 0.5 μM. Both Azacytidine and Decitabine were added to the 0.2x WGP bacterized media. The culture media was changed daily, and the cells were treated for four consecutive days until the cells reached 20 vegetative cycles, making them be able to go through autogamy.

ABT-199 and WEHI-539 were purchased from Selleck Chemicals (Breda, The Netherlands), A-1155463 and AZD5991 from Chemietek (Indianapolis, USA). 5-Aza-2′-deoxycytidine (Decitabine) and Oxaliplatin were purchased from Sigma Aldrich. All compounds were dissolved in DMSO to a stock of 20 mM, except Oxaliplatin, which was dissolved in water to a stock of 12.5 mM.