Deletion of Fbw7 leads to defective HSC quiescence and self-renewal. (A) LSK control and Mx1-cre⁺Fbw7^f/f (CKO) mice were further analyzed for DNA content (DAPI) and intracellular Ki-67 expression (2 wk after polyI-polyC). Quadrants correspond to cell cycle stages as shown in the left panel. The percentage of LSK cells in G0 stage is also shown (n = 6), (B) BrdU labeling of the CD34⁻ and CD34⁺ LSK subsets (n = 4) (2 wk after polyI-polyC). (C) 25,000 total BM cells from control or CKO mice were plated in methylcelluose medium containing a multilineage cytokine cocktail. The number of colonies and their qualitative morphologies were determined 8 d later. (D) The same experiment as in C was performed, except plating 500 flow-purified LT-HSCs per plate. After 7 d, the colonies were counted (first plating). The colonies from each plate were pooled and resuspended in PBS, and 2,000 of those cells were replated for an additional 7 d before counting colonies again (second plating). Numbers indicate the percentage of cells in each gate. (E) Photographs of representative colonies from C (first plating) show the relative size difference between control- and CKO-derived colonies. Bar, 0.5 mm. These are representatives of at least three individual experiments. *, P < 0.05; **, P < 0.005.