BRAF is dispensable for BCR-ABL–mediated ERK activation and transformation in vitro and in vivo. (A) Verification of the pMmiR^BRAF–BCR-ABL vector. Ba/F3 cells were retrovirally infected with indicated vectors and BCR-ABL, BRAF, Raf1, p-ERK, ERK, and actin levels were determined by Western blot analysis. (B) Results of methylcellulose colony formation assays. Murine BM cells were retrovirally infected with pMmiR^Ctrl–BCR-ABL, pMmiR^Raf1–BCR-ABL, or pMmiR^BRAF–BCR-ABL and plated in methylcellulose medium without growth factor supplement in the absence or presence of imatinib as indicated. Representative photographs of methylcellulose colonies of pMmiR^Ctrl–BCR-ABL, pMmiR^Raf1–BCR-ABL, or pMmiR^BRAF–BCR-ABL–transduced BM cells are shown (top panel) and CFUs were quantified 10 days after plating (bottom panel). Results shown are from 1 of 3 independent experiments performed in duplicates. P values were determined by Student t test. (C) Functional analysis of pMmiR^BRAF–BCR-ABL in murine primary BM. BM of 5-FU–pretreated donor mice was transduced with indicated vectors, sorted by FACS, and analyzed by Western blot. (D) BRAF knockdown has no effect on BCR-ABL–mediated leukocytosis in a BM transplantation model for CML. Mice were transplanted with pMmiR^Ctrl–BCR-ABL (n = 5), pMmiR^Raf1–BCR-ABL (n = 4), or pMmiR^BRAF–BCR-ABL (n = 5) transduced BM and WBC counts and (E) proportion of leukemic cells in peripheral blood were determined. (F) Kaplan-Meier plot detailing survival times of mice transplanted with pMmiR^Ctrl–BCR-ABL (n = 10), pMmiR^Raf1–BCR-ABL (n = 9) or pMmiR^BRAF–BCR-ABL (n = 10). Data from 1 of 3 independent experiments are shown.