Figure 4.

Functional contribution of each pathway to survival and differentiation. (A) One-thousand LSK Flk2⁻ cells were sorted per well from BM isolated from uninduced control, Pten, β-cat^Act, and Pten:β-cat^Act mice and cultured in ST medium (see the Materials and Methods). Within 12 h of sorting, OHT was added to the cultures for a final concentration of 1 μM. Cultures are depicted at 48 h after in vitro induction. (B) Cultures from A were stained with Sytox Green and Annexin V and analyzed by FACS. Representative FACS plots distinguishing live (Sytox Green-negative) from dead (Sytox Green-positive) cells. Live cells were further gated for Annexin V-positive (apoptotic) cells. Numbers within gates represent the mean ± SD from three independent experiments. (C) Cultures are as described in A at 4 dpi. (D) Absolute number of LSK cells and early hematopoietic progenitors in control and Pten mutant BM (tibia and femur) and spleen as determined by FACS analysis at 9–10 wpi. (E) Absolute number of LSK cells and early progenitors in the spleen as determined by FACS analysis on mice transplanted with control, β-cat^−/−, Pten, and Pten:β-cat^−/− BM as indicated; analysis is at 9–10 wpi.