FOXO proteins bind to the Hes1 promoter region and antagonize NOTCH target genes transcription. (A) Quantitative RT-PCR analysis of Nrarp and Hes1 in flow-sorted LSK cells and CMPs from FoxO^−/− or FoxO^+/+ animals. All signals were normalized to Gapdh and are shown relative to FoxO^+/+ RNA. Bar graphs represent the mean ± SEM of triplicate experiments. (B) RNAs from flow-sorted FoxO^−/− or FoxO^+/+ LSK cells and CMPs were extracted after 3 days of coculture with OP9-DL1 stroma and analyzed by quantitative RT-PCR as in panel A. Bar graphs represent the mean ± SEM expression normalized to Gapdh and shown relative to FoxO^+/+ cells grown on OP9-DL1 (n = 3). (C) ChIP of FoxO^+/+ or FoxO^−/− Lin⁻ cells with the use of an anti-FOXO1 or an anti-FOXO3a Ab. Anti-Histone H3 and anti-IgG Abs were used as positive and negative controls, respectively. Bar graphs represent the mean ± SEM of triplicate experiments, and all Hes1 or Nrarp promoter region signals are normalized to FoxO^+/+ sample pulled down with anti-Histone H3 Ab. (D) ChIP analysis of Lin⁻ cells infected with an empty MIG vector or with a wild-type FoxO3a construct and pulled down with an anti-FOXO3a Ab. All signals are normalized to MIG cells pulled down with an anti-IgG Ab, and bar graphs represent the mean ± SEM of triplicate experiments.