Figure 3.

T₃/TR represses Bim protein expression via downregulation of FoxO1. (a) RNA from TR-overexpressing or control J7 cells in the absence or presence of T₃ for 24 or 48 h was prepared prior to qRT-PCR analysis of FoxO1 mRNA expression. Values (means±S.E.M.) are shown as fold induction, compared with 0 nM T₃. All assays were repeated at least three times (**P<0.01). (b and c) FoxO1 protein levels in total lysates of isogenic J7 and Hep3B cell lines maintained in the absence or presence of T₃ (1 or 10 nM) for 24 or 48 h, determined using western blotting. ACTIN signals served as the loading control. (d) After transfection of GFP-FoxO1 for 48 h in J7 cells, the expression levels of FoxO1 and three isoforms of Bim were examined using western blotting. (e) Following ectopic expression of control, FoxO1-wt or FoxO1-AAA via adenoviral infection in J7-TR cells, the effects of T₃ on Bim protein expression were examined using western blotting. (f) Following T₃ (10 nM) stimulation for the indicated times, lysates of J7-TR cells were extracted for examining phosphor-AKT expression with western blotting. AKT1 and ACTIN were used as internal controls. (g and h) After T₃ (10 nM) treatment for the indicated times, cytoplasmic and nuclear fractions of J7-TR or GFP-FoxO1-overexpressing J7-TR cells were extracted for detecting endogenous FoxO1 or exogenous GFP-FoxO1 protein expression