Figure 6. 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.

(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 p53AIP1 and PUMA 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 TP53 knockdown on apoptosis induction by DS-7423. TP53 was knocked down by two independent siRNAs specific to TP53 (siRNA1 and 2) in OVISE cells, which do not carry any mutation in TP53. The apoptotic cell population was evaluated using annexin-V staining, as described in Figure 5. 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 TP53 knockdown on cell proliferation by DS-7423 in MTT assay of OVISE cells. TP53 was knocked down by a siRNA1 specific to TP53 and MTT assay was subsequently performed as in Figure 2. 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 TP53. 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.