Block in T cell development at DN1 stage in knockdown Mib1 in OP9-DL1 cells. (A) Immunoblot of Mib1 protein in OP9-DL1 cells 36 h after microporation with control (ctrl) or Mib1 siRNA. Dll1 expression was not affected. (B) Control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells were cocultured with C2C12-Notch1 cells transfected with 8× WT CBF-Luc vectors. 24 h after coculture, luciferase activities were measured. Data are mean ± SD from triplicate experiments. *, P < 0.001. (C) Lin⁻Sca-1⁺c-Kit⁺ fetal liver (FL-LSK) or BM (BM-LSK) cells were prepared and cultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. Growing cells collected on days 5 or 7, respectively, were stained for CD44 and CD25 gated on CD4⁻CD8⁻ DN cells and analyzed by flow cytometry. A representative of three independent experiments is shown. (D) C2C12-Notch1 cells were cocultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. After 12 h, the cells were stained with Dll1 antibody. Bars, 10 μm.