Drug-Induced Differentiation Figure 4. Nucleoside drugs cause differential DNA methylation changes in NT2 cells. (A) COBRA analysis of four CpG islands in the HOXA cluster in drug-treated and control NT2 cells, showing the demethylating activity of DAC and DZNep. ‘‘U’’ indicates unmethylated epialleles; ‘‘M’’ indicates methylated epialleles. (B) Genomic cytosine methylation levels decrease in DAC- and DZNep-treated NT2 cells. All measurements were repeated at least three times, standard deviations are indicated by error bars. P-values (Student’s t-test) for the differences between control and treated cells are shown on top. Only treatment with DZNep (DZN) and DAC (columns marked with an asterisk) lead to highly significant changes (p#0.01). (C) DNMT1 trapping assay in NT2 cells showing depletion of DNMT1 only after DAC-treatment. DNMT1 in drug-treated and control cells was detected by Western blot analysis of protein extracts from drug-treated cells. b-actin is shown as a loading control. (D) COBRA analysis of the same CpG islands as in (A) in DZNep- and DAC-treated cells, control cells and cells depleted for SAHH and DNMT1, showing that depletion of SAHH also leads to demethylation. ‘‘U’’ indicates unmethylated epialleles; ‘‘M’’ indicates methylated epialleles. Knockdown efficiencies for this experiment are shown in Figure S3C. doi:10.1371/journal.pone.0010726.g004 induced by araC (Figure S4B). No such effect could be observed for the reduction of OCT4 upon RA-treatment (Figure S4C), which is consistent with the notion that OCT4 is downregulated on the RNA level in RA-treated NT2 cells. DZNep has been shown to trigger proteasomal degradation of PRC2 components [14]. We thus also employed the proteasome inhibitor Lactacystin [37]. We observed no an effect on araC- or DAC-induced expression of HOXA1 or SNAP25 (Figure S6A) and Lactacystin did not rescue araC-induced OCT4 or EZH2 degradation (Figure S6B and S6C). In contrast, DZNep-induced EZH2 degradation was affected by Lactacystin (Figure S6C), which is in line with the established role of DZNep in inducing proteasome degradation [14]. The rescue effects exerted by caspase and JNK inhibitors on araC-induced differentiation could also be observed morpholog- ically (Figure 7B). Combinatorial treatments with araC and caspase inhibitors led to clearly less differentiated cells compared to araC-treatment alone. Similar effects were observed when CASPASE-3 and CASPASE-7 were depleted by corresponding siRNAs in araC-treated cells (Figure S7A). NT2 cells induced to differentiate with araC showed significantly less differentiated cells when CASPASE-7 or both caspases were knocked down after 6 hours of araC-treatment and subsequent incubation for 48 hours (Figure S7A). In addition, combined knockdown of CASPASE-3 and -7 in araC-induced NT2 cells led to significantly lower activation of HOXA1 and SNAP25 (Figure S7B) and also partially restored OCT4 protein levels in araC-treated NT2 cells (Figure S7C). In a final set of experiments we also tested if active recombinant human CASPASE-7 or CASPASE-3 is able to degrade OCT4 or NANOG in nuclear extracts of NT2 cells in vitro. As shown in Figure 7C, treatment with increasing amounts of CASPASE-7 led to a significant reduction of OCT4 signals, whereas similar treatment with CASPASE-3 had no effect on OCT4 stability PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10726