Activation of FOXO1 was necessary for DOX-induced Bim transcription.
A–C, NRCMs were treated with DOX (1 μm) in the presence of the FOXO1 inhibitor AS1842856 (1 μm) or vehicle for 4 h. A and B, protein levels were measured by Western blotting. C, mRNA levels were analyzed by quantitative RT-PCR. Two-way ANOVA with Sidak test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, NRCMs were transfected with control (siControl) or FOXO1 siRNA (siFOXO1) prior to treatment with DOX (1 μm) for 4 h. Bim mRNA levels were analyzed by quantitative RT-PCR. Two-way ANOVA with Sidak test. **, p < 0.01. E, H9c2 cells were transfected with the Bim promoter construct Bim-LUC or Bim-LUC(dm) with mutations in the FOXO1-binding sites. Cells were then treated with DOX (1 μm) for various periods of time, and luminescence was measured. One-way ANOVA with Tukey test. **, p < 0.01; ***, p < 0.001 versus time 0.