The effect of differentiation-inducing drugs in NT2 cells can be rescued by caspase and JNK inhibitors.

OCT4 is a substrate of CASPASE-7. (A) Quantitative RT-PCR expression analysis of the differentiation markers SNAP25, HOXA1 and of CASPASE-7 after 3 days of treatment with araC, DAC or RA alone or in combination with 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). 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. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding lamin-b and β-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 shown. Asterisks indicate expression values that are significantly different from controls. (B) Microscopic images (10× magnification) of NT2 control cells (cont.) and NT2 cells treated for 72 h with 1, 3 and 10 µM araC (upper panels) and NT2 cells co-treated for 72 h with a mixture of all three inhibitors (lower panels) are shown. The araC-induced neuron-like morphology can be reverted to some degree by co-treatment with caspase and JNK inhibitors. (C) Western blots showing the levels of OCT4 and NANOG in nuclear extracts from NT2 cells without treatment (cont.), in nuclear extracts incubated at 37°C alone in the presence of 1 (+) or 2 (++) units of active recombinant human CASPASE-7 or CASPASE-3. β-actin was used as loading control. Antibodies specific for N-terminal peptides of OCT4 and NANOG were employed. OCT4 signals are efficiently reduced only by CASPASE-7, whereas NANOG is degraded by both caspases.