FIG. 5.
FKHR-L1 directly regulates p27KIP1 transcription. (A) Expression of FKHR-L1 in Ba/F3 cells or Ba/F3 cells stably expressing FKHR-L1(A3):ER* was verified by immunoblotting with FKHR-L1 antibody. (B) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were electroporated with 12 μg of p27KIP1 luciferase construct together with 8 μg of pSG5. Cells were cultured with IL-3 (CON) or with IL-3 and 4-OHT (100 nM), and luciferase activity was analyzed 24 h later as described in Materials and Methods. (C) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were treated with 4-OHT (100 nM) for the indicated times; 20 μg of total RNA was used for Northern blotting and hybridized with a p27KIP1 probe (top). Equal RNA loading was verified by GAPDH reprobing (bottom). (D) Ba/F3 cells and Ba/F3 cells stably expressing FKHR-L1(A3):ER* were cytokine starved overnight and were cultured with IL-3 or with IL-3 and 4-OHT (100 nM). Equal amounts of protein were loaded, and the levels of p27KIP1 (top) and RACK1 (bottom) were determined by immunoblotting as described in Materials and Methods. (E) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were treated with 4-OHT (100 nM) in the absence or presence of actinomycin D (5 μg/ml) for the indicated times and analyzed as for panel D. (F) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were cultured in the absence or presence of IL-3 or IL-3 together with various concentrations 4-OHT overnight and were analyzed as for panel D.