The c-myc IRES was activated by GM-CSF and IL-3. (A) Schematic illustration shows dicistronic reporter gene constructs. The control plasmid pRF contains a short linker sequence between Renilla (Rluc) and firefly (Fluc) luciferase genes, and pRMF contains the c-myc IRES between 2 luciferase genes. phpRMF contains the c-myc 5′ untranslated region (UTR) between 2 luciferase genes and palindrome sequence upstream to Renilla luciferase gene. This palindrome sequence allows the formation of a stable hairpin in the RNA. Transcriptions of the 3 constructs were driven by SV40 promoter. (B) G1-arrested MO7e and F36P cells were transfected with pRF or pRMF by electroporation. After the incubation of cells with 10 ng/mL (MO7e) or 1 ng/mL (F36P) GM-CSF for 6 hours at 37°C, Renilla and firefly luciferase activities were determined, and IRES activities were expressed as ratios of firefly to Renilla luciferase. Horizontal axis represents the relative luciferase activity, which was calculated as relative to pRF, which was given a value of 1. (C) Elutriated G1-arrested cells were cotransfected with pSV-β-gal (used as control) and dicistronic plasmid (pRMF or phpRMF) and were then incubated with or without 10 ng/mL (MO7e) or 1 ng/mL (F36P) GM-CSF and IL-3 for 6 hours at 37°C. Both firefly (Fluc, upper panels) and Renilla (Rluc, lower panels) luciferase activities were normalized to β-galactosidase activity, and luciferase activity of stimulated cells was expressed as fold activation to that of unstimulated cells. Data presented are the means (± SEM) of duplicate samples from 3 independent experiments (B-C).
