DN1s from Miz-1^ΔPOZ mice lack in vitro differentiation and/or survival signals on OP9DL1 cocultures. (A) Comparative differentiation kinetics of 500 cells sorted from DN1, DN2, and DN3 subsets after 6 days of culture on stromal OP9DL1 cells. Gated live cells (top panel) were further gated on CD4⁻CD8⁻TCR-γδ⁻ and analyzed for CD44 and CD25 surface expression to assess DN stages of differentiation (middle panel). CD4 and CD8 surface expression shows developmental progression of more mature T cell (bottom panel). The numbers in dot plots are percentages of cells (n = 6). (B) Cell cycle analysis after in vivo BrdU labeling. Cells were stained for surface markers and anti-BrdU and gated on DN1, DN2, and DN3. Data show percentage of BrdU-positive cells (n = 2). (C) Cell cycle analysis using propidium iodide staining was performed on permeabilized, sorted DN1, DN2, and DN3 cells. Data show percentage of cells in S/G₂/M phase and are representative of 4 independent experiments. (D) Single-cell suspensions of thymocytes were stained with antibodies against lineage markers, CD44 and CD25 followed by annexin V staining. Percentages of annexin V-positive cells are indicated (n = 4).