Fig. 2.

Enhanced colony formation ability and leukemia initiation potential of PKH26 retention leukemia cells. (A) Colony formation ability of PKH⁺ and PKH⁻ leukemia cells. PKH⁺ and PKH⁻ cells were sorted from GFP⁺ bone marrow cells of PKH26‐stained leukemia cells transplanted mice on day 25 after transplantation and colony formation assay was then performed. Data are presented as the mean ± SEM of three independent experiments. The statistical significance was determined using unpaired t‐test. (B) Experimental schema for PKH26 staining and the serial transplantation assay. Equal number of sorted PKH⁺ or PKH⁻ cells was used in both the first round and the second round of transplantation. (C) Kaplan–Meier survival curves of primary recipients inoculated with PKH⁺ or PKH⁻ leukemia cells (PKH⁺: n = 7, PKH⁻: n = 7). The statistical significance of survival was determined using Log‐rank (Mantel–Cox) test. (D) Kaplan–Meier survival curves of the second transplantation (PKH⁺: n = 7, PKH⁻: n = 5). Transplanted PKH⁺ or PKH⁻ leukemia cells were sorted from secondary round recipients. For the second round of transplantation, GFP⁺ cells from primary leukemia mice transplanted with PKH⁺ cells were sorted and re‐stained with PKH26, and then injected into sublethally irradiated recipients. When these recipients developed leukemia, PKH⁺ and PKH⁻ cells were sorted and second transplantation was performed. The statistical significance of survival was determined using Log‐rank (Mantel–Cox) test.