Figure 4 - Effects of CGK733 on kinase signaling pathways related to ATM and ATR.

(a) Effects of CGK733 on a panel of kinases that can phosphorylate p53. CGK733 concentrations (left to right) were 0, 2, 4, 8, 16 and 32 M. (b) Effects of CGK733 on activation-associated phosphorylation of ATM and p53 and upregulation of p53 and p21 in cells with TRF2^BM-induced senescence, as determined by immunoblot analysis. (c,d) Effects of CGK733 on the nuclear focus (NF) and nuclear body (NB) formation associated with TRF2^BM-induced senescence, as detected by immunostaining with antibodies against phospho-ATM(Ser1981) or phospho-p53(Ser15), respectively. DNA was counterstained with Hoechst 33342. Scale bar, 20 m. Quantitative data are shown in d; >200 cells were scored. (e) Effects of CGK733 on ATR kinase activities inside cells. After incubation of U2OS cells with 5 M CGK733 for 4 h, these cells were stimulated with 2 mM hydroxyurea (HU) or 5 g ml^-1 aphidicolin (Aph) for 24 h or 2 h, respectively, and analyzed by immunoblotting. (f) Effects of CGK733 on PI3K activity in vitro, as determined by kinase assays with purified PI3K in the absence or presence of CGK733 or LY294002. (g) Effects of CGK733 on PI3K activity in live cells. BJ cells were transfected with the expression plasmid for PH-EGFP, incubated in a serum-reduced medium (0.5% FBS) for 40 h, and then treated with LY294002 (25 M) or CGK733 (100 M) 2 h before stimulation with 20 ng ml^-1 PDGF. After stimulation with PDGF, spatial distribution of PH-GFP was monitored by confocal microscopy. Scale bar, 100 m. (h) Effects of CGK733 on intracellular Akt activation. BJ cells were incubated in a serum-reduced medium (0.5% FBS) for 40 h and then treated with LY294002 or CGK733 (25, 50, and 100 M) 2 h before stimulation with 20 ng ml^-1 PDGF. After stimulation with PDGF for 10 min, the cells were analyzed by immunoblotting. (i) Effects of siRNAs against ATM and ATR on the proliferation of senescent cells. BJ cells with TRF2^BM-induced senescence were transfected with the siRNAs indicated, and cell numbers and protein levels were determined after 3 d. Averages of three experiments are shown; error bars, s.d. Protein levels of ATM and ATR were quantified and normalized with respect to actin.