NPM-ALK regulates JunB via mTOR and MAPK signaling. (A) NPM-ALK induces activation of AKT, ERK, and mTOR. Whole-cell lysates from vector control or NPM-ALK–expressing BA/F3 cells were immunoblotted with antibodies against proteins indicated on the right. (B) JunB protein expression is attenuated in NPM-ALK–expressing cells by mTOR or MEK inhibition. SR-786: NPM-ALK–positive ALCL cell line (top panel); TB-NA: TonBaF.1-NPM-ALK cells 24 hours after induction with 2 μg/mL doxycycline (+ dox), or without doxycycline (− dox) (bottom panel). Western blot analysis was performed with antibodies indicated on the right after treatment for 24 hours with 20 nM rapamycin, 12.5 μM U0126, both agents, or control (DMSO). (C) JUNB mRNA is decreased by MEK but not mTOR inhibition. qRT-PCR was performed on SR-786 cells and doxycycline-induced TB-NA cells upon treatment as indicated (20 nM rapamycin, 12.5 μM U0126, both agents, control [DMSO]). Logarithmic scale of mean JUNB expression levels related to expression of a housekeeping gene (β-actin) as 2^−ΔCT (bars indicate SEM of triplicate experiments). (D) Following 24-hour treatment with 20 nM rapamycin, 12.5 μM U0126, both agents, 48.8 μM LY294002, or control (DMSO), whole-cell lysates from SR-786 cells were blotted with antibodies against proteins indicated on the right. (E) Cell counts (normalized to control at t = 0) at indicated time points: Treatment conditions as in panel D resulted in reduced cell growth.