Activation of the PI3K pathway by BCR-ABL is required for elevated levels of ROS. Intracellular ROS levels were measured by DCF-DA staining (A-C). (A) BCR-ABL-transformed BaF3 cells were treated for 4 hours with wortmannin (10 nM) or rapamycin (1 nM) and compared with control-treated cells (top). (B) Parental BaF3 cells and cells expressing either BCR-ABL wild type (WT) or BCR-ABL containing a Tyr177Phe substitution (Y177F) were used and left untreated or stimulated with IL-3 (top). Representative FACS plots of the data are presented in the bottom panel; the top 3 curves represent cells treated with IL-3; the bottom 3, untreated. (C) Three individual clones of starved BaF3.TetON.myr-p110PI3K cells were either left untreated or treated for 18 hours with doxycycline (1 μg/mL). The changes in relative ROS levels were determined (top), and p85 PI3K expression or phosphorylation on specific serine and threonine residues in AKT and S6K was detected as indicated (bottom). The error bars indicate the standard errors of the mean. ^*Significant differences (P < .05) were observed between treated and control cells (n = 3).