Figure 2.

T₃/TR-dependent suppression of the Bim promoter via FoxO1. (a) The Bim 5′-flanking region (−1950 to +338; P1), Bim 5′-deletion and FoxO1 consensus-binding site mutants (P1–P8) were cloned into pGL3-basic-TK vector to generate luciferase reporter plasmids. The promoter region in these mutants is shown (left). After co-transfection with β-galactosidase (a transfection efficiency control), J7-TR cells were harvested and luciferase activity was measured following treatment with T₃ (0 or 10 nM) for 48 h. Luciferase activity was normalized to that of β-galactosidase (**P<0.01; *P<0.05). (b) The structure of the human Bim 5′-flanking region (positions −1950 to +338; P1) containing two putative FoxO1 consensus-binding sites. (c) Luciferase activities of the Bim promoter were analyzed in J7 cells after ectopic expression of control or FoxO1-wt via adenoviral transfection. Luciferase activity was normalized to that of β-galactosidase. Data represent means±S.E.M. of values derived from three independent experiments (**P<0.01; *P<0.05). (d) Following ectopic expression of control, FoxO1-wt or FoxO1-AAA via adenoviral (Ad-control, -FoxO1-wt or -FoxO1-AAA) infection in J7-TR cells, the effects of T₃ on Bim promoter (positions −1950 to +338; P1) activity were examined as for panel (a)