KLF4 promotes survival and self-renewal in leukemia stem/progenitor cells. (A) Cell cycle analysis of GFP⁺ LSK cells from Klf4^fl/fl and Klf4^Δ/Δ CML mice was performed by flow cytometric detection of DNA content at 15 dpt. Statistical analysis of phases of the cell cycle (fl/fl, n = 5; Δ/Δ, n = 4) is also shown (right panel; data are mean ± standard deviation [SD]). (B) Cell death was determined by flow cytometric detection of annexin V in purified GFP⁺ LSK cells from Klf4^fl/fl and Klf4^Δ/Δ CML mice 15 dpt after incubation for 24 hours (n = 4, mean ± SD). (C) Immunoblot analysis of PARP cleavage in GFP⁺ LSK cells purified from Klf4^fl/fl and Klf4^Δ/Δ CML mice. (D) Transplantation of a 1:1 mixture of wild-type (WT; CD45.1⁺.2⁺) and Klf4^Δ/Δ (CD45.2⁺) GFP⁺ LSK cells into lethally irradiated B6.SJL (CD45.1⁺) mice. (E) Flow cytometric analysis of donor-derived cells in peripheral blood at 15 dpt and 24 dpt, as described in (E) (n = 5, mean ± SD). (F) GFP⁺ LSK cells purified from Klf4^fl/fl or Klf4^Δ/Δ CML mice were transplanted into cytoablated mice to evaluate self-renewal. Kaplan-Meier survival curves of CML mice (fl/fl, n = 7; Δ/Δ, n = 3). (G) GFP⁺ LSK Flt3⁻ cells were plated in methylcellulose cultures. (H) GFP⁺ LSK CD150⁺ cells were serially replated in methylcellulose to assess colony formation and annexin V staining in each plating (fl/fl, n = 5; Δ/Δ, n = 5, mean ± SD). Data are representative of 2 or 3 independent experiments. *P < .05, **P < .01, ***P < .001, 2-tailed Student t test; P < .01, log-rank test. PI, propidium iodide.