Defective Notch activation in the Mib1-null microenvironment leads to an MPD. (A) Potential of Mib1-inactivated primary BM stromal cells to readily activate Notch signaling. The wild-type and mutant stromal cells were cocultured with C2C12-Notch1 cells transfected with the 8× wild-type and mutant CBF-Luc vectors. Twenty-four hours after coculture, luciferase activity was measured. The 8× mutant CBF-Luc lacks the CBF-binding sites and was used as a control. (B,C) Intact Notch activation in LSKs in the Mib1-null microenvironment. Lethally irradiated 4.5-week-old WT (n = 3) and MT (n = 3) mice were injected intravenously with 5 × 10⁶ BM cells from transgenic notch reporter mice.⁹  At 6.5 weeks after transplantation (mutant recipient mice suffered from an MPD), LSKs from reconstituted BM cells were analyzed by flow cytometry (B) and immunocytochemistry with cleaved Notch1 antibody (Val1744, in red) (C). Numbers indicate the distribution of GFP-negative (left rectangle) and -positive (right rectangle) LSKs. Scale bar: 10 μm. (D) Semiquantitative RT-PCR analysis of Notch/Notch ligands in cultured primary BM stromal cells. β-Actin was used for normalization. (E,F) Survival rate (E) and flow cytometric analysis of blood cells (F) from the reconstituted mice. Lethally irradiated 7- to 9-week-old CD45.2 MMTV-Cre;Mib1^+/f (WT, n = 10), MMTV-Cre;Mib1^f/f (MT, n = 20), MMTV-Cre;Rosa-Notch1 (N1ICD, n = 10), and MMTV-Cre;Mib1^f/f; Rosa-Notch1 (MT;N1ICD, n = 12) mice were injected intravenously with 5 × 10⁶ CD45.1 BM cells. Flow cytometric analysis of each BMT recipient mouse 7 weeks after transplantation (F). Numbers indicate the mean plus or minus SD (n = 5).