HOXA1 promoter region (IPHOXA1prom_up ACTGGAAAGT- TGTAATCCTATG; IPHOXA1prom_lo AGAAAGTTGGCA- CAGTCACG) in 10 ml PCR reactions, using the Absolute QPCR SYBR Green Mix (Themo Scientific) and a Roche LightCycler 480. PCR conditions: 1 cycle: 95uC615 minutes; 42 cycles: 95uC615 seconds/60uC640 seconds, read; melting curve 65uC– 95uC, read every 1uC. Cycle threshold numbers for each amplification were measured with LightCycler 480 software and enrichments were calculated as percentage of the input. Caspase assay Activity of CASPASE-3 and -7 in DZNep-, araC-, DAC- and RA-treated NT2 cells was assayed with the Caspase-Glo 3/7 Assay System (Promega) according to the instructions of the manufac- turer. P-values were calculated using Student’s t-test. Preparation of nuclear extracts and caspase treatment Nuclei from NT2 cells were prepared according to Wu [55]. The nuclear pellet was resuspended in lysis buffer (15 mM Tris-HCl pH 7.4, 150 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT and Roche complete protease inhibitor cocktail; 1 ml per 106 starting cells) and lysed by adding ammonium sulfate from a 4 M stock (pH 7,6) to a final concentration of 0.4 M for 15 minutes on ice. The lysate was centrifuged 30 minutes at 4uC and max. speed in a tabletop centrifuge and the supernatant was precipitated with solid ammonium sulfate (0.3 g per ml of supernatant). Proteins were pelleted at 4uC and max. speed in a tabletop centrifuge for 15 minutes. The protein pellet was dissolved in caspase buffer (50 mM Hepes pH 7,2, 50 mM NaCl, 0,1% CHAPS, 10 mM EDTA, 5% glycerol and 10 mM DTT), 10 ml per 106 starting cells. Aliquots were stored at 280 uC or directly used for caspase digestion. Aliquots corresponding to roughly 15 mg of total protein were treated for 20 minutes with 1 or 2 units of active recombinant human CASPASE-7 or CASPASE-3 (BioVision Research Prod- ucts) in a total volume of 20 ml at 37uC. SDS-PAGE loading buffer (86) was then added and the samples were separated on a 12% SDS-PAGE gel and transferred to nitrocellulose. Proteins were immunodetected using specific antibodies against NANOG (abcam ab21624), OCT4 (abcam ab18976, N-terminal peptide), OCT4 (abcam ab19857, C-terminal peptide) and b-actin (abcam ab8226). Histone preparation Pellets of NT2-cells were washed once with cold PBS and then lysed on ice in cold lysis buffer (10 mM Tris pH 6.5, 5.2 g/l sodium bisulfite, 1% Triton-X100, 10 mM MgCl2, 486 g/l sucrose, 1,2 g/l sodium butyrate). Lysates were centrifuged for 5 minutes at 4uC, max. speed in a tabletop centrifuge. Pellets were washed 36 with cold lysis buffer and once with wash buffer (10 mM Tris-HCL pH 7.4, 13 mM EDTA). Final pellets were resuspended in 100 ml water, 1 ml H2SO4 was added and the samples were incubate on ice for 1 hour. Then, precipitates were pelleted and the supernatants were transferred to new tubes. Isolated histones were precipitated by adding 10 volumes of acetone at 220uC over night. Precipitates were collected and pellets were dissolved in water. After Bradford assay the histone solution was set to 1 mg/ml, stabilised by adding PAGE loading buffer to 16 and stored at 220uC. Generation of cell lines expressing OCT4-specific EGFP Roughly 16106 NT2 cells were transfected with the plasmid phOCT4-EGFP, containing a 4 kb fragment of the human OCT4 Drug-Induced Differentiation regulative region (23917 to +55) upstream of EGFP [35], using the Effectene Transfection Kit (Qiagen) according to the manufacturer’s instructions. Cells were plated into 10 cm culture dishes and G148 (Sigma) selection was applied 24 hours after transfection at 350 mg/ml. After 2 weeks of selection surviving colonies were picked and expanded. One clone, showing approximately 90% of EGFP positive cells, was chosen for treatment with araC and RA. Supporting Information Figure S1 Nucleoside drugs affect the viability of MCF7, NT2, HeLa and A549 cells in a similar manner. Cells were treated with increasing concentrations of DZNep, araC, DAC (40 nM up to 100 mM) and viability was analysed via resazurin reduction after 3 days. Error bars represent standard deviations; deoxycytidine (dC) was included as a mock treatment control. Found at: doi:10.1371/journal.pone.0010726.s001 (5.78 MB TIF) Figure S2 Effects of drug-treatment on EZH2-stability. DZNep- mediated EZH2 depletion is not caused by SAHH inhibition. (A) Western blots showing drug-induced degradation of EZH2 in the cancer cell lines MCF7, A549, HeLa, HEL and HL60. Total extracts from cells treated with DZNep, araC and DAC for three days were analysed. b-actin was used as loading control. Below the blots intesities of the EZH2-bands, normalised to the correspond- ing b-actin bands, are indicated as percentage of the control (cont.). (B) Western blot showing the effect of SAHH depletion (kd SAHH) on EZH2 and H3K27me3 levels in MCF7 cells. Cells were treated for 72 hours or 2 times 48 hours with siRNAs. Knock down of EZH2 (kd EZH2) was used as positive control; b-actin was used as a loading control. A scrambled siRNA was used as negative control (kd cont.). Reduction of SAHH-levels has no effect on EZH2 stability or H3K27me3 methylation. Found at: doi:10.1371/journal.pone.0010726.s002 (5.42 MB TIF) Figure S3 Effects of drug-treatment on HOX expression in NT2 cells. Efficiency of RNAi. Effect of RA-treatment on EZH2- stability. (A) qRT-PCR expression analysis of HOXA1, HOXA2 and HOXA3 in NT2 cells after 3 days of drug incubation. Drug- treatment at increasing concentrations (40 nM up to 20 mM) was done for 3 days and corresponding total RNA was reverse transcribed and analysed. Expression of all three HOX genes increases with drug concentration. Y-axis values indicate fold induction compared to the non-treated control. Deoxycytidine (dC) was included as a mock treatment control. All qRT-PCR measurements were repeated at least three times and internally normalized to the corresponding lamin-b and b-actin expression levels. Error bars represent standard deviations. (B) EZH2 transcription is efficiently reduced after siRNA mediated knock down in NT2 cells. EZH2 expression was quantified by qRT-PCR analysis in three independent knockdown experiments. Y-axis values indicate fold reduction compared to the scrambled control (kd cont.). (C) SAHH and DNMT1 transcription is efficiently reduced after siRNA mediated knock down in NT2 cells. Expression was quantified by qRT-PCR analysis in the knock- down experiment used for the COBRA analysis shown in figure 4D. Y-axis values indicate fold reduction compared to the scrambled control (kd cont.). (D) Western blot showing the effect of RA-treatment on the stability of EZH2 in NT2 cells during one month of treatment. Reduced protein levels could be observed after 3 weeks. b-actin was used as loading control. Found at: doi:10.1371/journal.pone.0010726.s003 (5.08 MB TIF) Figure S4 Differentiation-inducing drugs deplete NANOG and OCT4 by caspase-dependent degradation in NT2 cells. (A) PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10726