(A) Binding activity of soluble swapping chimeras based on Dll1 or Dll4 to Notch1 or Notch2 expressed on the surface of BM-derived mesenchymal cells with or without the glycosylation by lunatic or manic fringe (Lfng, Mfng) is measured by flow cytometry. Soluble Dll chimeras, depicted in Figure 3A, are composed of the extracellular region of Dll1, Dll4, or their chimeras and Fc domain of human IgG1. Soluble Dll1 (green), D1-D4DSL (red), D1-D4DOS (blue), and D1-D4DSL/DOS (orange), shown in the right side of panels, were prepared and incubated with Notch1 transfectants. Their bindings were detected by anti-human IgG Ab and shown in the first lane of upper panels. Soluble Dll4 (green), D4-D1DSL (red), D4-D1DOS (blue), and D4-D1DSL/DOS (orange) were also used in the second lane of upper panels. Similarly, soluble Dll-derived molecules were used to check their binding activities to the Notch2 transfectants with or without glycosylation in the lower panels. (B) Binding activity of the DSL/EGF1-2 swapping chimeras with soluble Notch1 and Notch2. Soluble Notch receptors are composed of the N-terminus to 15th EGF repeats and the Fc domain of human IgG1. The transfectants expressing Dll1, Dll4, or their chimeras were incubated with soluble Notch1 (sNotch1) or Notch2 (sNotch2), and their binding were detected. The soluble Notchs were produced by CHO cells expressing murine Lfng (red line), Mfng (blue line), or vector control (green line), and used in this experiment. (C) Induction of T-lineage cells by the swapping chimeras in vitro. Fetal liver-derived lineage markers-negative c-kit-positive hematopoietic progenitors were cultured on the OP9 cells expressing Dll1, Dll4, or their chimeras for 13 days. After the cultures, live cells were stained with CD19, Thy1, CD4, and CD8, and analyzed by flow cytometry. Numbers in the dot-plot represent the relative percentages for each corresponding quadrants.