Effect of STAT5b-RARα and other APL fusion proteins on the STAT3 transcriptional and DNA binding activity.

(A) Transactivation activities of STAT5b, STAT5b-RARα, RARα, PML-RARα, and PLZF-RARα in HepG2 cells. HepG2 cells were transiently transfected with 500 ng of APRE-luciferase reporter gene, 500 ng of β-galactosidase expression vector, and 2.0 μg STAT5b, STAT5b-RARα, RARα, PML-RARα, and PLZF-RARα. Transfected cells were stimulated with IL-6 (25 ng/mL) for 24 hours. Luciferase activity was measured and normalized for transfection efficiency using a β-galactosidase reporter construct. Data represent the mean ± SD of 5 separate experiments. The luciferase activity shown in lane 1 is increased 120-fold over the activity of identical cells incubated without IL-6 (not shown). (B) Gel-shift assays with WCEs from HepG2 cells transiently transfected with STAT5b or STAT5b-RARα. Transfected cells were incubated without or with IL-6 (25 ng/mL) for 30 minutes. The APRE (upper panel) and the hSIE (middle panel) were used as duplex oligonucleotide probes in this study. The location of the specific STAT3/DNA complex (upper panel and middle panel) and STAT5b/DNA (upper panel, lane 5) are indicated by the solid triangle and arrow, respectively; the empty triangle indicates the location of a nonspecific band. Levels of protein expression in the transiently transfected cells were determined by immunoblotting with the antibody against the N-terminal region of STAT5b (bottom panel).