Fig. 2.

Both KCC-07 and DNA damage induction result in known p53 dependent gene expression. (A) Induction of CDKN1A expression, which is p53 dependent and involved in cell cycle regulation, by either KCC-07 or DNA damaging reagent treatments measured by real-time PCR. Combined treatment with KCC-07 and DNA damaging reagents did not further increase CDKN1A expression in both cell lines. The SH-SY5Y cell line is more sensitive to DNA damaging reagents. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (B) Induction of BBC3 expression, which is p53 dependent and involved in apoptosis, by DNA damaging reagents. KCC-07 treatment did not induce BBC3 expression in both cell lines. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (C) Consistent DNA damage response (DDR) following combined treatment with KCC-07 and DNA damaging reagents analyzed by Western blots. Both cell lines displayed well-established DDR including ATM phosphorylation, KAP1 and CHK2 phosphorylation (ATM-dependent), and p53 activation (ATM-dependent) followed by induction of apoptotic and cell cycle arrest factors (p53-dependent, see B and C in this figure) upon DNA damage induction by Phleo or ETP treatments. KCC-07 addition does not change the proper DDR upon DNA damage, but sustained H2AX phosphorylation indicates defective DNA damage repair. Ponceau S staining and GAPDH western blot served as loading controls. Representative data are shown. Each experiment was repeated twice independently. The experimental conditions of drug treatments are indicated in the figure. NS, not significant.