Figure 4
BCR–ABL with NUP98–HOXA9 augments Evi1 expression to cause CML-BC in vivo. (a) Experimental scheme of Evi1-reporter CML-BC mice. 5FU-primed Evi1+/GFP BM cells with retroviral BCR–ABL and NUP98–HOXA9 were transplanted into lethally irradiated recipient mice. (b) GFP intensities of LK cells and BM cells from Evi1-reporter CML-BC mice were analyzed by FCM (upper left). The difference of Gr-1-positive rate in BCR–ABL (+) BM cells (upper right) and that of c-kit-positive rate in BCR–ABL (+) Lin– BM cells (lower) were shown. (c) The proportion of leukemic blasts in Evi1-high or Evi1-low CML-BC BM cells was shown (n=3). (d) Evi1-high or Evi1-low cells from CML-BC BM cells were sorted, cytospun and subjected to Wright–Giemsa staining. (e) GFP-positive rates of stem/progenitor fractions and BM cells in Evi1-reporter CML-BC or CP mouse were shown (n=13 for CML-BC mice, n=5 for CML-CP mice). (f) Semisolid colony assay was done with 1000 Evi1-high or Evi1-low CML-BC LK cells (n=5). (g) BCR–ABL mRNA expression was measured for each subset from CML-BC mice (n=3). (h) Representative FCM plot of LK cells from CML-BC mice (red) and LSK cells from CML-CP mice (blue). GFP intensity of normal BM cells is shown in solid gray. (i) Cell cycle status of Evi1-high or Evi1-low LK cells of CML-BC mice. Hoechst 33342 was measured by FCM (n=3). Data are mean±s.d. *P<0.01, **P<0.001, ***P<0.0001.
