Serial transplantation experiments with Arf^–/– BM. (A) Experimental design for serial transplantation experiments. Primary recipients received 2 × 10⁵ cells of either BL6, Arf^–/–, 129/BL6, or Ink4a-Arf^–/– BM cells. After 10 weeks, these primary recipients served as transplant donors for secondary recipients. Four groups of secondary recipients were given transplants at the indicated cell doses. Each group of recipients consisted of 5 to 10 animals. For subsequent transplantation cycles, the tertiary and quaternary recipients received the same dose of cells as was used for the donor, as shown in the numbers above the arrows. (B) Survival of secondary recipients of transplants with 1 × 10⁵ BM cells either from Arf^–/– BM cells (▴) or from wild-type control cells (□). (C) Analysis of Arf^GFP/+ allele expression after transplantation. Primary recipients received 2 × 10⁵ cells of either BL6 wild-type or Arf^GFP/+ BM cells. During the reconstitution phase of the transplantation (1.5, 2.5, and 3.5 weeks after transplantation), BM was harvested, stained with c-kit-APC, Sca1-PE, and Lin-APC-cyanine 7 (Cy7) antibody cocktail, and analyzed by flow cytometry for GFP expression in different subpopulations, including the KSL fraction. Shown is a representative graph of GFP levels in KSL fraction 2.5 weeks after transplantation; the 1.5- and 3.5-week time points also indicated absence of the GFP expression in KSL or other subfractions. Gates for GFP-positive cells and percentage of gates cells are shown.