Successful generation of leukemic HPC^LSKcell lines with various oncogenes. (A) Experimental design: HPC^LSK cell lines were retrovirally transduced with different oncogenes. (B) Immunoblot showing increase of CRKL, FLT3, JAK2, STAT5, ERK, and AKT phosphorylation and upregulation of cABL, c-MYC, and p53 in transformed HPC^LSK cells compared with untransformed (−) cells to the corresponding oncogenes. HSC70 serves as a loading control. Representative blot from at least 3 independent experiments is shown. (C) Flow cytometry analysis of untransformed and BCR/ABL^p210 transformed HPC^LSK cells in IMDM/SCF/IL-6 and SCF/IL-6 deprived medium (IMDM). After 1 month in culture, HPC^LSK BCR/ABL^p210 cells show reduced expression of stem cell markers (c-Kit, Sca-1) and differentiate into myeloid (CD11b, Gr-1), but not lymphoid (CD19, CD3) cells as indicated by the numbers in quadrants. The data are expressed as mean ± SD of 3 independent measurements. (D) Representative flow cytometry plots of LSK (upper), myeloid (middle), and lymphoid staining (lower) of MLL-AF9 (in the presence of SCF and IL-6), Flt3-ITD;Nras^G12D, and BCR/ABL^p185 transformed HPC^LSK and pre-pro-B BCR/ABL^p185 cell lines in the absence of SCF and IL-6.