GM-CSF and IL-3 induced c-myc IRES activity in another factor-dependent cell line UT7, in which c-myc mRNA was regulated by GM-CSF. (A) Cell cycle analysis of UT7 cells with or without 1 ng/mL GM-CSF. Determination of cell cycle distribution was performed as described in the legend to Figure 1. The G1 cells indicated by the open box were separated by centrifugal elutriation for the following experiments. (B) Elutriated G1-arrested UT7 cells were incubated with 1 ng/mL GM-CSF for the indicated periods at 37°C. Analyses of mRNA and protein were performed as described in the legend to Figure 1. (C) UT7 cells were labeled with radioisotope for 30 minutes at 37°C and were then incubated for the indicated periods with or without 1 ng/mL GM-CSF or IL-3 at 37°C. Determination of the half-life of the c-Myc protein was performed as described in the legend to Figure 2. Asterisk indicates nonspecific band. (D) The effect of rapamycin on GM-CSF– and IL-3–induced c-Myc expression in UT7 cells. Elutriated G1-arrested UT7 cells were pretreated with diluent (DMSO) or 250 nM rapamycin 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 subjected to Western blotting as in the legend to Figure 3. (E) Determination of GM-CSF– and IL-3–induced c-myc IRES activity in UT7. Elutriated G1-arrested UT7 cells were cotransfected with pSV-β-gal and dicistronic plasmid (pRMF or phpRMF) and were then incubated with or without 1 ng/mL GM-CSF or IL-3 for 6 hours at 37°C. Luciferase assay was performed as in the legend to Figure 4. Data presented are the means (± SEM) of duplicate samples from 3 independent experiments.
