Raf1 depletion attenuates BCR-ABL–induced transformation of murine BM cells in vitro. 5-FU–enriched mouse BM-derived progenitor cells were infected either with pMmiR^Ctrl–BCR-ABL or pMmiR^Raf1–BCR-ABL retrovirus. (A) Expression levels of Raf1 were analyzed by qRT PCR (TaqMan) in EGFP⁺ cells 5 days after transduction. Results were normalized to the housekeeping gene GAPDH. (B) Raf1 knockdown efficacy was verified by Western blot. (C) Retrovirally infected BM cells were starved for 6 hours in RPMI medium containing 3% FCS and no cytokines and p-ERK status was determined by Western blot. (D) Results of methylcellulose colony formation assays. PMmiR^Ctrl–BCR-ABL and pMmiR^Raf1–BCR-ABL–expressing murine BM cells were plated in methylcellulose without growth factors in the absence or presence of imatinib as indicated. Representative photographs of methylcellulose colonies of pMmiR^Ctrl–BCR-ABL or pMmiR^Raf1–BCR-ABL–transduced BM cells are shown and CFUs were quantified 10 days after plating. Results shown are from 1 of 3 independent experiments performed in duplicates. P values were determined by Student t test.