Induction of the phosphorylation of TP53 at Ser46 and the accumulation of transcripts of the genes targeted by TP53, which participate in TP53-mediated apoptosis.

<p>(A) Immunoblotting in OVMANA cells treated with DS-7423 at the indicated doses. Phosphorylation levels of MDM2 were inversely associated with p-TP53 at Ser46, but not with p-TP53 at Ser15. (B) Semi-quantitative RT-PCR in OVMANA and OVISE cells treated with DS-7423 at the indicated doses. Both <i>p53AIP1</i> and <i>PUMA</i> were induced by DS-7423. CT, untreated (negative) control; IR, irradiation at 14 Gy (positive control). GADD45 was induced in OVISE, but not in OVMANA cells. (C) Quantification of the semi-quantitative RT-PCR in (B). Each experiment was repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (D) Effect of <i>TP53</i> knockdown on apoptosis induction by DS-7423. TP53 was knocked down by two independent siRNAs specific to <i>TP53</i> (siRNA1 and 2) in OVISE cells, which do not carry any mutation in <i>TP53</i>. The apoptotic cell population was evaluated using annexin-V staining, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087220#pone-0087220-g005" target="_blank">Figure 5</a>. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (E) Suppression of TP53 expression by siRNAs was confirmed by immunoblotting. (F) Effect of <i>TP53</i> knockdown on cell proliferation by DS-7423 in MTT assay of OVISE cells. TP53 was knocked down by a siRNA1 specific to <i>TP53</i> and MTT assay was subsequently performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087220#pone-0087220-g002" target="_blank">Figure 2</a>. Knockdown of TP53 diminished the anti-proliferative effect caused by DS-7423 on OVISE cells. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (G) TP53 expression plasmid (0.1 µg/µL) was cotransfected with pp53 TA Luc (0.25 µg/mL) plasmid into ES-2 cells mutated in <i>TP53</i>. The addition of DS-7423 increased the relative luciferase activity of TP53 in a dose-dependent manner. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05.