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Fig. 4. BEL induced the phosphorylation of p53 in ATM^+/+, ATM^–/– cells and U2OS cells through doxycycline-stimulated expression of ATR-wt or ATR-kd. (A) Comparison of BEL-induced phosphorylation of p53 in GM01805 (ATM^+/+) and GM01526 (ATM^–/–) cells. ATM^+/+ cells and ATM^–/– cells were treated with or without BEL (15 µM) for 4 or 8 hours. Cell lysates were analyzed for p53 and p53-P by western blotting. (B) Response to BEL in ATM^–/– (GM01526) cells over time. ATM^–/– cells were cultured in the absence or presence of caffeine (2.5 mM) for 12 hours and continuously cultured in the presence or absence of BEL (12.5 µM) for 30 minutes and 3 hours. Levels of p53-P and p53 were examined by western blotting. (C) BEL-induced phosphorylation of p53-P in ATR-wt-inducible U2OS cells. ATR-wt-inducible U2OS cells (GW33) were cultured with or without doxycycline (1 µg/ml) for 1 day. The expression of FLAG-ATR-wt was determined using an anti-FLAG monoclonal antibody. The cells were first cultured in the absence or presence of caffeine (2.5 mM) for 12 hours and then in the absence or presence of BEL (12.5 µM) for 8 hours. Cells were collected and the levels of FLAG-ATR-wt, p53-P, p53, PCNA and actin were examined by western blotting. (D) BEL-induced p53-P in ATR-kd-inducible U2OS cells. ATR-kd-inducible U2OS cells (GK41) were cultured with or without doxycycline (1 µg/ml) for 1 day. The expression of FLAG-ATR-kd was examined using an anti-FLAG monoclonal antibody. The cells were treated with increasing concentrations of BEL for 8 hours and collected for analysis of FLAG-ATR-kd and p53-P by western blotting.

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