Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells
Figure 2
Increased Proliferation of KrasG12D HSC
Bone marrow from 5-wk-old WT and KrasG12D mice was stained with 7-AAD and pyronin Y (PY) for DNA and RNA quantitation, along with surface markers for Flk2− LSK cells.
(A) Gating is shown for cell cycle analysis of WT and KrasG12D Flk2− LSK cells.
(B) Summary of replicate samples from WT (closed circles) and KrasG12D (open circles) mice (n = 3 or 4 as shown; compiled from two independent experiments). Means and SEM are WT: G0 81.8 ± 4.72, G1 5.66 ± 1.76, S-G2-M 11.7 ± 2.56; and KrasG12D: G0 43.6 ± 3.19, G1 34.9 ± 4.85, S-G2-M 21.4 ± 3.14. p-Values by unpaired t-test are indicated: ***, p < 0.001; **, p < 0.01; *, p < 0.05.
(C) RNA from doubly sorted Flk2− LSK cells was isolated, and quantitative PCR performed on cDNA to test expression levels of selected cyclins (n = 3 or 4 as shown; bar shows geometric mean). Results are expressed as fold change in expression compared to WT Flk2− LSK cells, after normalization to β-actin expression. These results represent three or four independent experiments as shown, each performed with pooled bone marrow from three to five animals. The geometric mean of cyclin D1 expression is 2.4-fold over WT (95% confidence interval 1.2–4.9). Purity of sorted cells is shown in Figure S1.