Activated Stat5 directly mediates the IL-3 induction of enhanced RAR transcriptional activity in EML cells.

(A) Uninduced EML cells were electroporated with the βRARE-tk-Luc reporter (25 μg) together with expression vectors (40 μg of each) for wild-type Stat5 (Stat5aWT) or the constitutively active Stat5 (Stat5aHS). Following overnight incubation, the cells were harvested and relative luciferase activity was determined on cell extracts using the β-galactosidase reporter (20 μg) as an internal control. (B) SCF-dependent EML cells were incubated overnight in the presence or absence of IL-3 (5 ng/mL) as indicated. The cells were then electroporated with the βRARE-tk-Luc reporter (25 μg) together with expression vectors for the indicated Stat5a constructs (40 μg of each). The electroporated cells were then cultured with or without IL-3 for an additional 5 hours, and relative luciferase activity was determined on cell extracts using a β-galactosidase reporter (20 μg) as an internal control. For panels A and B, results represent the averages and SDs from at least 3 independent experiments. (C) SCF-dependent EML cells were stimulated overnight with IL-3. These cells were electroporated with the βRARE-tk-Luc reporter (25 μg) together with the indicated amount of the different Stat5a expression vectors. The cells were then cultured for an additional 5 hours in IL-3 and the relative luciferase activity determined. Results represent the averages and SDs from triplicate samples.