Self-renewal of AML1-ETO9a-transduced bone marrow cells is impaired in absence of <i>Inca1</i>.

<p><b>A.</b> Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from <i>Inca1^+/+</i> or <i>Inca1^−/−</i> mice was retrovirally transduced with AML1-ETO9a-IRES-GFP, MLL-AF9-IRES-GFP, or c-myc-IRES-BCL2-IRES-mCherry. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses. <b>B.</b> Engraftment of AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells was determined by FACS analysis of GFP-positive cells in the blood of transplanted mice from 4 to 40 weeks. Engraftment was initially higher in recipients transplanted with <i>Inca1^−/−</i> bone marrow cells. The number of GFP-positive cells in <i>Inca1^−/−</i> bone marrow decreased significantly from weeks 4 and 8 until week 40 (*p = 0.015 and **p = 0.04, respectively). This analysis was restricted to mice without overt leukemia. <i>Inca1^+/+</i>: n = 14 at 4 and 8 weeks, n = 9 for 32 and 40 weeks; <i>Inca1^−/−</i>: n = 10 at 4 weeks, n = 9 at 8 and 32 weeks, n = 7 at 40 weeks. <b>C.</b> Colony assays with two subsequent replatings using AML1-ETO9a-positive <i>Inca1^+/+</i> or <i>Inca1^−/−</i> bone marrow cells, respectively, that were FACS-sorted from non-leukemic transplanted mice (n = 3 from each genotype). <i>Inca1^−/−</i> bone marrow cells transduced with AML1-ETO9a formed less colonies and replated worse than transduced <i>Inca1^+/+</i> cells (1^st plating: p = 0.01; 2^nd plating: p = 0.03; 3^rd plating: n.s.). <b>D and E.</b> For a cloning efficiency assay, 1 to 300 GFP-positive <i>Inca1^+/+</i>; AML1-ETO9a or <i>Inca1^−/−</i>; AML1-ETO9a bone marrow cells from non-leukemic transplanted mice were FACS-sorted two (<b>D</b>) or six months (<b>E</b>) and lin⁻GFP⁺ cells were seeded in semi-solid medium in a 48-well plate (n = 14 for each concentration). Two months after transplantation, <i>Inca1^+/+</i> cells had a clone forming frequency of 1/31, while the frequency was much lower in <i>Inca1^−/−</i> cells (1/164; p = 0.0001) (<b>D</b>). After six months, cloning efficiency of <i>Inca1^−/−</i> cells decreased to 1/483 (<b>E</b>).