Fig. 1

BRAF amplification is reversible in cells with acquired resistance to MEKi. a, b Non-clonal COLO205 cells with acquired resistance to selumetinib (C6244-R cells, R) and 12 single-cell clone derivatives of C6244-R (1–12) were treated with 1 μM selumetinib (Sel) (a) or selumetinib-free medium (b) for 24 h. Parental COLO205 cells (P) were treated in parallel with selumetinib-free medium for 24 h. Lysates were western blotted with the indicated antibodies. c, d Following 12.5 weeks culture in the presence (+) or absence (COLO205 and (−)) of 1 μM selumetinib, cells were treated with the indicated concentrations (10 nM to 10 μM) of selumetinib (Sel) for 24 h, and DNA synthesis assayed by [³H]thymidine incorporation (c), or incubated in selumetinib-free medium for 24 h and lysates western blotted with the indicated antibodies (d). C6244-R C1 and C6244-R C2 are single-cell clonal derivatives of C6244-R. Results (c) are mean ± SD of cell culture triplicates and normalized to control for each cell line. e, f Following 10 weeks culture in the presence (+) or absence (HT29 and (−)) of 1 μM selumetinib, cells were treated with the indicated concentrations (10 nM to 10 μM) of selumetinib (Sel) for 24 h, and DNA synthesis assayed by [³H]thymidine incorporation (e), or incubated in selumetinib-free medium for 24 h and lysates western blotted with the indicated antibodies (f). HT6244-R C1 and HT6244-R C2 are single-cell clone derivative cell lines of HT6244-R. Results (e) are mean ± SD of cell culture triplicates and normalized to control for each cell line. g, h Following 20 weeks culture in the presence (+) or absence (COLO205 and (−)) of 1 μM selumetinib (g), or 10 weeks culture in the presence (+) or absence (HT29 and (−)) of 1 μM selumetinib (h), BRAF locus PAC DNA (RP5-1173P7; green) and chromosome 7 centromere probe (red) were hybridized to metaphase spreads and interphase nuclei (grey, DAPI). C1 and C2 are single-cell clonal derivatives of C6244-R or HT6244-R as indicated. Scale bars indicate 2 µm (metaphase panels) and 10 µm (interphase panels)