1 mM DAC, 20 mM araC, 20 mM dC and 5 mM DZNep (if not otherwise indicated). Substances, from aqueous stock solutions (5 or 50 mM), were added once to freshly seeded cells (26105 in a 6- well culture plate in 2 ml of medium). Treatment with JNK Inhibitor 1 (Alexis Biochemicals, 1 mM stock solution in water) at 1 mM, caspase Inhibitor I and II (Calbiochem, 5mM stock solution in DMSO) at 5 mM and with the proteasome inhibitor Lactacystin (Sigma L6785, 5 mg/ml stock solution in water) at 2 mM was done 2 hours before araC, DAC and RA were added. Inhibitors were then added every day. Cells were harvested by trypsin treatment and stored at 280uC. Viability assay Cell viability was assayed using the CellTiter-Blue Cell Viability Assay (Promega). NT2, MCF7, HeLa or A549 cells were seeded into 96-well assay plates at a density of 1000 cells per well in 100 ml of medium. Test compounds araC, DAC, DZNep and dC were added in increasing concentrations from 40 nM up to 100 mM and cells were grown for 72 hours. Assays were performed in 6 biological replicates. After 3 days 20 ml of CellTiter-Blue reagent was added to each well, mixed and incubated for 1–4 hours. Absorbance at 570 and 600 nm was measured for each well and resazurin reduction was calculated according to the instructions of the kit. Viability was plotted as percentage compared to the untreated cells. RT PCR and real time analysis Total RNA from drug-treated and untreated NT2 tissue culture cells was prepared using the Trizol-reagent (Invitrogen) according the manufacturer’s specifications. Real-time RT-PCR was per- formed using the QuantiTect Reverse Transcription Kit (Qiagen). One mg of total RNA was processed in 20 ml reactions according to the manufacturer’s specifications. One ml of the cDNA was used for a 10 ml PCR reaction using the Absolute QPCR SYBR Green Mix (Thermo Scientific) and a Roche LightCycler 480. PCR conditions: 1 cycle: 95uC615 minutes; 42 cycles: 95uC615 se- conds, 60uC640 seconds, read; melting curve 65uC–95uC, read every 1uC. Cycle threshold numbers for each amplification were measured with LightCycler 480 software and relative quantifica- tions of expression status of the genes under observation comparing treated to non-treated cells were calculated and normalised using lamin-b and b-actin as internal standards. qRT- PCR measurements were repeated at least three times on biological replicates. P-values were calculated using Student’s t- test. For primer sequences see Table S1. COBRA assay For methylation analysis of CpG-rich regions in the HOXA cluster genomic DNA was treated with sodium bisulfite and analysed by COBRA [53]. PCR cycling conditions were 95uC for 30 seconds, 55–60uC for 30 seconds, and 72uC for 40 seconds for 35 cycles. The PCR products were gel purified and digested with BstUI or TaqIa (New England Biolabs, Beverly, MA). Digested PCR products were separated on 3% agarose gels. For primer sequences see Table S2. Capillary electrophoresis Genomic DNA was prepared from cells using the DNeasy Kit (Qiagen). Global methylation levels were determined by capillary electrophoresis, as described previously [54]. Briefly, genomic DNA was enzymatically hydrolysed to single nucleotides and the nucleotides were derivatised with the fluorescent marker BODIPY (Molecular Probes). Derivatised nucleotides were separated by Drug-Induced Differentiation capillary electrophoresis and analysed in a Beckman PACE MDQ Molecular Characterization System. P-values were calculated using Student’s t-test. DNMT1 trapping assay NT2 D1 cells were grown and treated with the indicated concentrations of DZNep, araC and DAC as described above. After 3 days of treatment, cells were harvested, washed with PBS and lysed for 30 minutes on ice in trapping buffer (PBS pH 7.4, 0,1% SDS, 0,5% NP40, 0,5% Sodiumdeoxycholate, 1 mM DTT and Roche complete protease inhibitor cocktail). Insoluble debris was pelleted and supernatants were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Milli- pore). DNMT1 was detected using an antibody from Santa Cruz Biotechnologies (C-17, sc-10222). Protein depletion via siRNAs For depletion of SAHH, DNMT1, EZH2, NANOG and OCT4 ON-TARGETplus SMARTpool siRNAs (Dharmacon) were used. NT2 cells were seeded into 6-well plates at a density of 26105 in 2 ml of medium. siRNAs were transfected with the Dharma- FECT1 transfection reagent (Dharmacon) according to the manufacturer’s instructions. The final siRNA concentration was 50 nM. For combined knock downs the reactions were scaled up accordingly. After 48 h or 72 hours cells were retransfected (48 h), analysed by microscopy or harvested by trypsin treatment. Cell pellets were then analysed by Western blot and RT-PCR. Scrambled siRNAs for negative control experiments were also obtained from Dharmacon. For depletion of araC-induced CASPASE-3 and/or CASPASE-7 cells were treated for 6 hours with 10 mM araC and were then transfected with scrambled siRNAs, CASPASE-3 specific siRNAs, CASPASE-7 specific siRNAs and a mix of both as described above. After transfection the cells were grown for further 48 hours (without changing the medium), analysed by microscopy, then harvested by trypsin treatment and analysed by Western blot and RT-PCR. Western blotting and antibodies Pellets of cultured cells were lysed in SDS-PAGE-buffer, shock frozen in liquid nitrogen and heated for 5 minutes at 95uC. Samples were separated on 8–10% SDS-PAGE and transferred to nitrocellulose (Whatman) or polyvinylidene difluoride (Millipore) membranes. Proteins were immunodetected using specific anti- bodies against EZH2 (Cell Signalling AC22), NANOG (abcam ab21624), OCT4 (abcam ab18976), b-actin (abcam ab8226) and SAHH (obtained from Michael Hershfield, Durham). Histones were separated using 15% SDS-PAGE gels and transferred to nitrocellulose (Whatman) membranes and immunodetected using specific antibodies against H3 (Cell Signaling 9715), H3K27me3 (Upstate/Millipore 07-449) and cH2AX (Milipore, clone JBW301). In some cases (Figures 2A and S2A) intensities of protein bands were background corrected and quantified using imageJ software (available at: http://rsb.info.nih.gov/ij/), nor- malised against the actin loading control and calculated as percentage of the signal for the untreated control. ChIP assay Crosslinked chromatin was prepared and immunoprecipitated as described previously [9]. ChIP-grade antibodies specific for H3K27me3 (07-449) and H3K4me3 (07-473) were purchased from Upstate. Immunoprecipitates were finally dissolved in 30 ml of TE buffer (10 mM Tris-HCL pH 8, 1 mM EDTA). One ml was analysed by real time PCR using a primer pair specific for the PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10726