Characterization of OP9 stromal cells expressing different human Notch ligands. (A) Flow cytometric analysis of OP9-control, OP9-DLL1, OP9-DLL4, OP9-JAG1, or OP9-JAG2 stromal cells for EGFP expression. (B) Flow cytometric analysis of EGFP and Notch ligand expression in OP9-DLL1, OP9-DLL4, and OP9-JAG2 cells, as indicated. Isotype and OP9-control stainings were used as negative controls, and ligand specificity was determined through the absence of antibody staining on OP9 cells that express a different Notch ligand. (C) Flow cytometric analysis of Notch ligand expression in OP9-DLL1, OP9-DLL4, and OP9-JAG2 cells (black histograms), as indicated, versus OP9-control cells (gray histograms). (D) Differentiation of Notch1-expressing C2C12 myoblast cells (C2C12N1) after 5-day coculture with OP9-control, OP9-DLL1, OP9-DLL4, OP9-JAG1, or OP9-JAG2 in the presence of 1μM GSI or 0.1% dimethyl sulfoxide as a control. Arrows indicate myotube formation. Images were acquired from cells in culture medium with a Leica DM IL microscope (20×/0.3 objective) using a Leica DCF420 camera with Leica 3.1.0 Application Suite software.