Figure 2. Nucleoside drugs affect EZH2 stability in NT2 cells. (A) Western blot showing EZH2 in control (cont.) cells and cells treated with dC, DZNep (DZN), araC, DAC and RA for 3 days. Reduced EZH2-levels were observed for araC, DAC and DZNep. b-actin was used as loading control, deoxycytidine (dC) as a mock treatment control. Below the blots intensities of the EZH2-bands, normalised to the corresponding b- actin bands, are indicated as percentage of the control (cont.). (B) EZH2 mRNA expression levels in NT2 cells upon 3 days of drug treatment do not change significantly, as determined by quantitative RT-PCR. qRT- PCR measurements with at least three biological replicates were internally normalised to the corresponding lamin-b and b-actin expression levels. Deoxycytidine (dC) was included as a mock treatment control. (C) DZNep-mediated EZH2 depletion is not caused by SAHH inhibition. Western blot showing the effect of SAHH depletion (kd SAHH) on EZH2 and H3K27me3 levels in NT2 cells. Cells were treated for 72 hours with siRNAs. Knockdown of EZH2 (kd EZH2) was used as Drug-Induced Differentiation positive control, b-actin and histone H3 (H3) were used as loading controls. A mix of scrambled siRNAs was used as negative control (kd cont.). Reduction of SAHH-levels has no effect on EZH2 stability or H3K27me3 levels. doi:10.1371/journal.pone.0010726.g002 inhibition of S-adenosylhomocysteine hydrolase. Our results thus confirm the demethylating activity of DZNep and suggest that DZNep-induced DNA demethylation is not related to DNMT1 degradation. Nevertheless, the lack of detectable araC-induced methylation changes suggests that inhibition of DNA methylation is not required for HOXA derepression and cellular differentiation. Drug-induced differentiation of NT2 cells requires degradation of stem cell factors In our previous experiments we found differentiation by RA, araC and DAC to be accompanied by a loss of PcG-repression at the HOXA-cluster. However, RA did not trigger EZH2-depletion and EZH2-degradation by DZNep did not lead to HOXA- activation. This suggests that the drug-induced loss of PcG repression is caused by an upstream event. A recent study described the CASPASE-3-dependent degradation of the stem cell-specific transcription factor NANOG as important for RA- induced differentiation of human ES cells [33]. NANOG and the core transcription factor OCT4 maintain the expression of stem cell genes, and repress the induction of differentiation-specific factors [5]. A reduction of NANOG and OCT4 protein levels is thus a prerequisite for the induction of differentiation. Both araC and DAC, but not DZNep [14], are incorporated into DNA and have been shown to induce DNA damage, apoptosis and proteolytic cascades [25]. Consistently, we observed a robust accumulation of the DNA-damage marker cH2AX in cells treated with araC and DAC (Figure 5A). As DNA-damage is generally a trigger for caspase activation [34,35], we next measured the enzymatic CASPASE-3/7 activity of drug-treated cells. Indeed, treatment with araC and DAC led to a substantial increase in caspase activity, while RA treatment had only a moderate effect and DZNep did not lead to clear caspase activation (Figure 5B). Of note, CASPASE-3/7 activation was paralleled by an effective depletion of NANOG and OCT4 (Figure 5C and S4A). DZNep had no effect on cH2AX, NANOG and OCT4, whereas RA-treatment did not lead to an increase of cH2AX, but also reduced both stem cell factors (Figure 5A, 5C and S4A). The lack of OCT4 reduction in DZNep-treated NT2 cells indicates that the mild morphological changes observed (Figure 1A) are not due to differentiation processes. Downregu- lation of NANOG and to lesser extent OCT4 by araC and DAC was observed also at the mRNA level, while RA led to a very pronounced transcriptional repression of both genes (Figure 5D). To further distinguish between protein degradation and regulation on the RNA level, we established a transfected NT2 cell line expressing EGFP under the control of the OCT4 regulatory region [36]. Untreated cells strongly expressed EGFP, whereas treatment with retinoic acid for three days led to a significant reduction of the EGFP-signal (Figure 5E), indicating that OCT4 expression is silenced on the mRNA level. Treatment with araC for the same period of time resulted in cellular differentiation, but the cells retained significant EGFP expression (Figure 5E), which is consistent with OCT4 downregulation at the protein level. In order to test if direct depletion of OCT4 and NANOG induces differentiation of NT2 cells, we reduced the protein levels of OCT4, NANOG, EZH2, DNMT1 and SAHH by dsRNA interference and monitored neuronal morphology and expression PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10726