Figure 5.

Sequential addition of AUY and SCH to OS cells. (a) OS1002 cells received 100 nℳ AUY or 10 nℳ SCH for 12 h (black bars). Untreated (gray bars) and pretreated cells were refed with fresh medium containing SCH, AUY or both and were harvested 36 h after refeeding. (b) OS1002 cells received 100 nℳ AUY for 6 or 12 h (black bars). Untreated (gray bars) and pretreated cells were refed with fresh medium containing SCH (10 nℳ) or SCH plus AUY and were harvested 36 h after refeeding. (c) Gray bars: OS1002 cells were refed with medium containing 10 nℳ SCH, 100 nℳ AUY or both and were harvested 48 h later. Black bars: OS1002 cells pretreated with AUY for 18 h were refed with fresh medium containing SCH or were incubated in medium alone for 1 or 4 h before addition of SCH. Cells were harvested 48 h after SCH addition. (a–c) Percent apoptotic cells was determined by FACS analysis of annexin V-stained cells. C: control. (d) OS1002 cells received 100 nℳ AUY for 16 h. Cells were refed with fresh medium with (+) or without AUY and were harvested at the indicated times. Amounts of AKT, phospho-AKT (pAKT) and actin (loading control) were determined by western blotting