c-Myc-deficient thymocytes differentiate without proliferation. FACS-sorted DN3 and DN4 cells from the indicated mice were labeled with CFSE and cocultured with OP9-DL1 cells for 4 days. Cell suspensions from the OP9-DL1 cocultures were stained with antibodies against CD4, CD8, or TCRαβ and analyzed by flow cytometry. Empty histograms represent results for LckCre Mycfl/fl; filled gray histograms represent results for LckCre control mice. (A) Top panels show 2-parameter dot plots of CD4 versus CFSE staining of the indicated subsets and mice. Bottom panels show histogram overlays comparing CFSE in LckCre versus LckCre-Mycfl/fl cells after the coculture. (B) Top panels show 2-parameter dot plots of CD4 versus CD8 surface staining of the indicated cells and mice. Numbers represent the percentage of total events in the shown gates. (C) TCRαβ surface expression in the gates shown in the 2-parameter dot plots. Arrows depict the starting populations used in the cocultures. In vitro differentiation was observed in 3 independent experiments.
