Western blots showing levels of NANOG and OCT4 proteins in cells treated for 6, 12, 24 and 48 hours with araC, DAC and RA. b-actin was used as a loading control. The blot showing retinoic acid induced changes (on the right) was reprobed after NANOG-staining with OCT4 antibodies. Thus the b-actin control is the same for this experiment. DAC- and araC-treated OCT4 and NANOG blots are from two different experiments. (B) Western blots showing that drug-induced degradation of OCT4 and EZH2 in NT2 cells after 36 hours (OCT4) and 72 hours (EZH2) of treatment with araC is reduced (the signal is restored) by caspase or JNK inhibitors. b-actin served as loading control. (C) Western blots showing that RA-induced reduction of OCT4 48 hours of treatment with retinoic acid is not significantly reduced by caspase inhibitors I and II (Casp-I, Casp-II), JNK inhibitor 1 (JNK), a combination of both Caspase inhibitors (Casp-I/II) and a mixture of all three inhibitors (all). b-actin was used as loading control. Found at: doi:10.1371/journal.pone.0010726.s004 (5.11 MB TIF) Figure S5 Depletion of stem cell factors induces differentiation of NT2 cells. (A) Microscopic images (106 magnification) of NT2 control cells (cont.) and NT2 transfected with a mix of scrambled siRNAs (kd cont.), and siRNAS specific for DNMT1 (D), SAHH (S), EZH2 (E), NANOG (N), OCT4 (O) and various combinations of these. After transfection the cells were grown for 72 hours. Knock down of OCT4 clearly induces neuronal phenotypes in NT2 cells. (B) Quantitative RT-PCR expression analysis of the differentiation marker SNAP25 and of DNMT1 (D), SAHH (S), EZH2 (E), NANOG (N) and OCT4 (O) after the knock down experiment described in (A). Diagrams show fold differences compared to control cells (cont.). The transcription of DNMT1, SAHH, EZH2, NANOG and OCT4 is efficiently reduced after the respective siRNA mediated knock down. Expression of the differentiation marker SNAP25 is weakly induced by NANOG knock down, but significantly upregulated upon depletion of OCT4, which is in line with the morphological observations. A combined knock down of NANOG and OCT4, or NANOG, OCT4 and EZH2 does not enhance the effect. qRT-PCR measurements are from two independent experiments and were internally normalised to the corresponding lamin-b and b-actin expression levels. P-values (Student’s t-test) for the expression differences between untreated cells (cont.) and transfected cells for highly significant cases (p#0.01) are indicated. Asterisks indicate expression values that are significantly different from controls. Found at: doi:10.1371/journal.pone.0010726.s005 (5.49 MB TIF) Figure S6 The effects of araC and DAC can not be rescued by the proteasome inhibitor Lactacystin. (A) Quantitative RT-PCR expression analysis of the differentiation markers SNAP25 and HOXA1 in NT2 cells after 3 days of treatment with araC, DAC or RA alone or in combination with the proteasome inhibitor Lactacystin (LAC) or a combination of two caspase inhibitors (Casp-I/II). Diagrams show fold differences compared to araC-, DAC- and RA-treatment alone. Co-treatment with caspase inhibitors leads to reduced gene expression after araC- and DAC-treatment, whereas Lactacystin had no significant effect. 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 (Student’s t-test) for the expression differences between only drug-treated and co-treated cells for highly significant cases (p#0.01) are indicated. Asterisks indicate expression values that are significantly different from controls. (B) Western blots showing that drug-induced degradation of OCT4 in NT2 cells after 36 hours of treatment with araC is reduced (the signal is restored) by the mix of caspase inhibitors (Casp), but not Drug-Induced Differentiation by treatment with Lactacystin (LAC, middle). b-actin served as loading control. (C) Western blots showing that drug-induced degradation of EZH2 in NT2 cells after 72 hours of treatment with araC is reduced (the signal is restored) by the mix of caspase inhibitors (Casp), but not by treatment with Lactacystin (LAC, middle). In contrast, DZNep-induced degradation of EZH2 is also inhibited by Lactacystin (blot on the right). b-actin served as loading control. Found at: doi:10.1371/journal.pone.0010726.s006 (4.70 MB TIF) Figure S7 The effect of differentiation-inducing drugs in NT2 cells can be rescued by depletion of CASPASE-3 and -7. (A) Microscopic images (106 magnification) of NT2 control cells (cont.) and NT2 cells treated for 54 h with 10 mM araC (araC). The remaining images show cells that were treated for 6 hours with 10 mM araC, then were transfected with a mix of scrambled siRNAs (kd cont.), CASPASE-3 specific siRNAs (kd CASP-3), CASPASE-7 specific siRNAs (kd CASP-7) and a mix of both (kd CASP-3/7). After transfection the cells were grown for further 48 hours (with araC present). The araC-induced neuron-like morphology can be reverted to some degree by the double knock down. (B) Quantitative RT-PCR expression analysis of the differentiation markers SNAP25, HOXA1 and of CASPASE-3 and CASPASE-7 after the treatment described in (A). Diagrams show fold differences compared to araC-treatment alone. CASPASE-3 and CASPASE-7 transcription is efficiently reduced after the respective siRNA mediated knock down. Combined knock down of both caspases leads to a significantly reduced araC- induced expression of SNAP25, HOXA1. qRT-PCR measure- ments are from two independent experiments and were internally normalised to the corresponding lamin-b and b-actin expression levels. P-values (Student’s t-test) for the expression differences between only araC-treated (cont.) and transfected cells for highly significant cases (p#0.01) are indicated. Asterisks indicate expression values that are significantly different from controls. (C) Western blots showing that araC-induced degradation of OCT4 (under the conditions described in A) is reduced (the signal is restored) by combined depletion of CASPASE-3 and CAS- PASE-7. b-actin served as loading control. Found at: doi:10.1371/journal.pone.0010726.s007 (4.08 MB TIF) Figure S8 Potential caspase sites in the OCT4 protein. (A) Predicted caspase substrate cleavage sites by the CASVM (server for SVM Prediction of Caspase Substrates Cleavage Sites - http:// casbase.org/casvm/index.html) using the P4-P29and P14-P109- trained classifiers are listed. The four amino acid core sequence (P4-P1) and the position of the P1-site along the 360 amino acid sequence of OCT4 are shown for each potential or predicted site. (B) The positions of the 5 predicted cutting sites are indicated below a sketch of the OCT4 protein. The POU-domain and the homeodomain are highlighted. The calculated molecular weight in kD for potentially originating N-terminal fragments after caspase digest are indicated below the arrows. The potential site used by CASPASE-7 is marked with an asterisk (C) Western blots showing the levels of OCT4 in nuclear extract from NT2 cells without treatment (cont.), in nuclear extract incubated at 37uC or treated with 1 (+) or 2 (++) units of active recombinant human Caspase-7 at 37uC. An antibody specific for an N-terminal peptide of OCT4 was used. b-actin was used as loading control. The blot is identical to the one shown in Fig. 7C but was exposed longer. A digestion product of ca. 30 kD becomes visible, that is significantly increased upon CASPASE-7 digestion (arrow with asterisk). This would correlate with a cut at the predicted site within the homeodomain of OCT4 (asterisk in B). Found at: doi:10.1371/journal.pone.0010726.s008 (5.86 MB TIF) PLoS ONE | www.plosone.org 13 May 2010 | Volume 5 | Issue 5 | e10726