CPT triggers p53 phosphorylation at Serine 46.

<p>(a) Time course of p53 phosphorylation at serine 46 upon CPT treatment was analyzed by Western blotting using anti-phospho p53 Ser46 (p53pSer46) specific antibody, immediately after (3h), 6h or 15 h post drug removal (3+6 h, 3+15 h, respectively). GAPDH was used as loading control (left panel). p21^Waf1 expression levels upon CPT exposure in hiPSCs and hESCs-derived NP were analyzed by Western blotting immediately after 1μM CPT treatment (3h), 6h or 15h post genotoxic removal (3+6 h, 3+15 h, respectively) using specific antibodies against p21^Waf1 and Actin (right panel) (b) mRNA levels of <i>bcl-2</i>, <i>bax</i> and <i>puma</i> were analyzed by Real Time RT-PCR in CPT treated or untreated hESCs, hiPSCs and NP. GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to undamaged hESCs arbitrarily set as 1. Each bar represents the mean ± SEM of three independent experiments. In all cases, a paired Student's t test was used to compare CPT-treated samples to untreated ones (c) Cell viability was measured by the XTT/PMS assay at 6 h after drug removal in PFT-μ treated or untreated cells (10 μM, 1 h before and during CPT exposure). Results are presented as the percentage of the viability of untreated cells (left panel). PFT-μ treated or untreated cells were harvested 3h after CPT treatment (1 μM over a 3 h period), and DNA oligomers were measured by immunoassay. Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean ± SEM of three independent experiments performed in triplicate (right panel) In all cases, a paired Student's t test was used to compare CPT plus PFT-μ treated samples to CPT treated ones.*P< 0.05, ***P< 0.0001.