Role of RAS and MAP kinases in MCL-1 expression in BCR/ABL-transformed cells. (A,B) Ton.B210-X cells were transfected with mcl-1-luc/pCMV-βGal, and in addition with either pcDNA3-ras-N17, pcDNA3-ras-G12V, or empty (pcDNA3) vector. Cells (nontransfected or transfected) were incubated with doxycycline (1 μg/mL) to induce BCR/ABL in the presence or absence of inhibitors, that is, PD98059 (50 μM) or STI571 (1 μM) as indicated, for 16 hours at 37°C in serum-free UltraCulture medium. After incubation, cells were harvested and assayed for luciferase and βGal activities. Luciferase activity was reported as the ratio mcl-1-luc/pCMV-βGal. Results are given as percent of control (ie, empty vector) and represent the mean ± SD of 3 independent experiments. (C) Ton.B210-X cells were grown in serum-free UltraCulture medium in the absence of doxycycline (control) or were induced to express BCR/ABL (BCR/ABL) by addition of doxycycline. After 15 hours, cells were exposed to PD98059, 50 μM (BCR/ABL + PD98059) for 45 minutes. Then, cells were harvested and subjected to Western blotting. Expression of phosphorylated ERK was determined using a pMAPK antibody (top row). After stripping, the membrane was reprobed with a MAPK antibody (bottom row). (D) K562 cells were cultured in serum-free UltraCulture medium containing STI571 (1 μM) for 16 hours, or in UltraCulture medium for 15 hours followed by incubation in PD98059 (50 μM) for 45 minutes. After incubation, cells were harvested and subjected to Western blotting. Expression of phosphorylated ERK (top row) and total ERK (bottom row) was determined using a pMAPK and a MAPK antibody, respectively.