Figure 2.

TGFβ inhibits nuclear translocation of exogenous FKHRL1 and Forkhead-dependent transcription. (A) Cells seeded in 60-mm dishes (10⁶ cells/dish) were transfected with 10 μg/dish of either WT- or TM-FKHRL1-HA for 16 h and transferred to coverslips on 12-well plates. The cells were then incubated in serum-free medium for 24 h followed by stimulation with 2 ng/ml TGFβ for 30 min with or without 20 μM LY294002. Fixation and staining were performed as described in MATERIALS AND METHODS. Fluorescence intensities associated with anti-HA fluorescein and Hoechst 33258 were observed with a Zeiss Aiophot upright microscope. (B) Cells were transfected with WT-or TM-FKHRL1, each with FHRE-Luc and pCMV-Rl vectors followed, where indicated, with removal of serum and the addition or not of 2 ng/ml TGFβ for 24 h. Dual luciferase assay was performed as described in MATERIALS AND METHODS. Relative luciferase units represents firefly luciferase activity normalized to Renilla luciferase activity. Each data point represents mean ± SD of four wells.