Figure 5.

APAP-induced Bim transcription is JNK- and Foxo3a-dependent. (a) IHH cells were stimulated with APAP (10 mM) or, as positive control, phorbol 12-myristate 13-acetate (30 ng/ml) and phospo-JNK and tubulin protein levels were analyzed at different time points by western blot. (b) IHH cells were transfected with the wild-type (WT) Bim reporter construct, pre-treated with JNK V inhibitor (10 μM) and stimulated with APAP (10 mM) or phorbol 12-myristate 13-acetate (30 ng/ml). (c) Similarly, IHH cells were transfected with the WT Bim reporter construct, pretreated with different concentrations of the JNK inhibitor V and stimulated with APAP (10 mM). Luciferase activity was measured and normalized to β-galactosidase. (d) IHH were pretreated with JNK inhibitor V and stimulated with APAP (10 mM). Bim mRNA expression was measured by quantitative RT-PCR (6 h). (e) IHH cells were treated as described above and Bim were analyzed at different time points by western blot. (f) IHH cells were transfected with empty vector (pGL2), the wild-type (Bim WT) or the Foxo3a mutant Bim reporter constructs (Bim Foxo3a), and stimulated with APAP (10 mM) for 6 h. Luciferase activity was measured and normalized to β-galactosidase activity. Mean values±S.D. of triplicates are shown for relative luciferase units and fold induction of mRNA levels. The experiments have been repeated three times, yielding similar results