Label-retaining cells were present in immortalized, premalignant cells in vitro. Immortalized, premalignant cell populations NSP2 (a, b), NSP3 (c, d), and SP3 (e, f) were cultured in vitro as described in Materials and methods. Cells were plated, allowed to incubate overnight, treated with 0.5 μM EdU for 24 hours, and fixed, permeabilized, labeled with Alexa Fluor® 488, and counterstained with DAPI either (a, c, e) immediately or (b, d, f) after serial passage. Representative images are shown. Scale bars equal 40 μm.