GSI-treated tumors do not appear to develop GSI resistance. Thymomas from MRK-003– or vehicle-treated mice were harvested from the animals and converted to in vitro culture. (A) Leukemic cell lines from vehicle- and GSI-treated mice express high levels of intracellular Notch1 and remain GSI responsive. Leukemic cell lines were treated for 48 hours with 1 μM MRK-003 or DMSO carrier. Cell lysates were examined for intracellular Notch1 levels by immunoblotting with an anti-Notch1^IC Val1744 (no. 2421; Cell Signaling Technology) and anti–β-actin antibodies. (B) Leukemic growth remains Notch1 dependent. Leukemic cell lines, generated from vehicle- and GSI-treated mice, were treated with vehicle or 1 μM MRK-003 for 3 and 6 days. Cells were then assayed for DNA content by staining with propidium iodide followed by flow cytometry. The figure is a representative experiment using cell line 6838.