Drug-Induced Differentiation Figure 5. Differentiation-inducing drugs cause DNA damage in NT2 cells and deplete NANOG and OCT4 by caspase- dependent degradation. (A) Western blots showing the levels of cH2AX in untreated NT2 cells (cont.) and after 72h treatment with the indicated drugs. Histone H3 was used as loading control. (B) Caspase-Glo-assay of NT2 cells treated for 48 hours with DZNep (DZN), araC, DAC and RA. CASPASE-3/7-activity is shown as direct luminescence. Error bars represent standard deviations of at least 3 independent experiments. P-values (Student’s t-test) for the differences between control and treated cells are shown on top. All observed changes are significant (marked with an asterisk). (C) Western blots showing the levels of NANOG and OCT4 in untreated NT2 cells (cont.) and after 72h treatment with the indicated drugs. b-actin was used as loading control. (D) Quantitative RT-PCR analysis of NANOG and OCT4 after 24, 48 and 72 hours of treatment with DZNep, araC, DAC and RA. Diagrams show fold differences compared to the untreated control. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding lamin-b and b-actin expression levels. P- values for the expression differences between control and treated cells for highly significant cases (p#0.01) are shown. Asterisks indicate expression values that are significantly different from controls. (E) Microscopic images (106 magnification) of NT2 cells expressing EGFP under the control of the OCT4-promoter (first row) and the same cell line cells treated with RA (second row) and araC (third row) for 3 days. The first column shows the light microscopic images (phase contrast), the second row EGFP fluorescence, the third column an overlay. EGFP- signals persist in araC-treated cells. doi:10.1371/journal.pone.0010726.g005 (Figure 7C). In contrast, NANOG is degraded under similar conditions by both caspases, with CASPASE-3 showing a significantly stronger effect (Figure 7C). These experiments confirm that OCT4 and NANOG are direct substrates for CASPASE-7 and support published data showing that NANOG is targeted by CASPASE-3 [30]. Taken together, we identified CASPASE-7 and, to a lesser extent, CASPASE-3 as important mediators of araC- and DAC- PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10726