Potent inhibitory effects of LY294002, but not rapamycin, on GM-CSF– and IL-3–induced Rb phosphorylation and cell cycle progression. (A) Elutriated G1-arrested cells were pretreated with diluent (DMSO), 250 nM rapamycin, or 50 μM LY294002 for 30 minutes at 37°C and were then incubated with or without 10 ng/mL (MO7e) or 1 ng/mL (F36P) GM-CSF and IL-3 for 3 hours (MO7e) or 9 hours (F36P) at 37°C. Total cell lysates (3 × 105 cells per lane) were separated by SDS-PAGE and were then immunoblotted using specific antibody for Rb. (B) Elutriated G1-arrested MO7e cells were pretreated with diluent (DMSO), 250 nM rapamycin, or 50 μM LY294002 for 30 minutes at 37°C and were then cultured with or without 10 ng/mL GM-CSF in the presence of 10 μM BrdU for 24 hours at 37°C. Detection of incorporated BrdU was performed as described in “Materials and methods.” (C) Elutriated G1-arrested MO7e cells were pretreated with diluent (DMSO), 250 nM rapamycin, or 50 μM LY294002 for 30 minutes at 37°C and were then cultured with or without 10 ng/mL GM-CSF for 24 hours at 37°C. Analysis of cell cycle distribution was performed as in the legend to Figure 1.
