Potent inhibitory effects of LY294002 on GM-CSF– and IL-3–induced protein and mRNA synthesis of c-myc, c-myc IRES activity, and Rb phosphorylation in UT7 cells. (A) Elutriated G1-arrested UT7 cells were pretreated with diluent (DMSO), 50 μM LY294002, or 50 μM PD98059 for 30 minutes at 37°C and were then incubated with 1 ng/mL GM-CSF or IL-3 for 2 hours at 37°C. Total cell lysates were separated by SDS-PAGE and analyzed by immunoblotting as described in the legend to Figure 5. (B) Elutriated G1-arrested UT7 cells were pretreated with diluent (DMSO) or 50 μM LY294002 for 30 minutes at 37°C and were then incubated with 1 ng/mL GM-CSF or IL-3 for the indicated periods at 37°C. Analysis of c-myc mRNA was performed as described in the legend to Figure 1. (C) Elutriated G1-arrested UT7 cells transfected with dicistronic luciferase plasmid (pRMF or phpRMF) and pSV-β-gal were pretreated with diluent (DMSO) or 50 μM LY294002 for 30 minutes at 37°C and were then cultured with or without 1 ng/mL GM-CSF or IL-3 for 6 hours at 37°C. Luciferase assay was performed as described in the legend to Figure 4. Data presented are the means (± SEM) of 3 independent experiments. (D) Elutriated G1-arrested UT7 cells were pretreated with diluent (DMSO), 250 nM rapamycin, or 50 μM LY294002 for 30 minutes at 37°C and were then cultured with 1 ng/mL GM-CSF or IL-3 for 9 hours at 37°C. Immunoblotting for Rb was performed as described in the legend to Figure 6.
