c-Myc ablation at the DN3 stage impacts thymic cellularity and subset distribution. (A-B) Efficiency of c-Myc ablation at the DN3 and DN4 stages of thymocyte development. Semiquantitative c-Myc RT-PCR with 5-fold serial dilutions (A) and c-Myc Western blots (B) were performed on FACS-sorted cells from the indicated mice and subsets. Data shown are representative for 3 independent experiments. (C) FACS analyses for CD4/CD8 (top) and CD44/CD25 (bottom, gated on lin- events) surface expression in LckCre-Mycfl/fl and LckCre (Adult = 5-8 weeks old) or Mycfl/fl (E 16 = Embryonic day 16) mice. Numbers given indicate the percentage of events in the respective quadrant. Data shown represent observations from more than 10 independent experiments (Adult) and 2 independent experiments (E 16). (D) Cellularity was determined by multiplying the number of total thymocytes with the percentages from panel A (for DN3, DN4 also considering the percentage of lin- cells). Error bars indicate SD; Thy, total number of thymocytes; and γδ, TCRγδ+ thymocytes. Numbers of adult animals analyzed to obtain these statistics were as follows (NLckCre-Mycfl/fl, NLckCre): total thymocytes (n = 27, n = 11), DP/CD4+/CD8+/DN (n = 13, n = 6), DN3/DN4 (n = 9, n = 7), and TCRγδ+ thymocytes (n = 10, n = 5). Embryo data are based on 3 Mycfl/fl control embryos and 11 LckCre-Mycfl/fl.
