Knockdown of JUNB impairs cell-cycle progression in NPM-ALK–expressing cells. (A) Small hairpin (sh) RNA knockdown of JUNB in doxycycline-induced TonBaF.1-NPM-ALK cells. (Right) JUNB mRNA is decreased in 24-hour doxycycline-induced TonBaF.1-NPM-ALK cells stably transduced with JUNB sh-RNA–producing vector (sh-JUNB) compared with cells producing scrambled sh-RNA (sh-control). JUNB expression levels of sh-control–treated cells were set as 1 to facilitate comparison with sh-JUNB cells (bars indicate standard deviation [SD] of triplicate experiments). (Left) Cell counts (normalized to control at t = 0) demonstrate a decreased cellular proliferation of sh-JUNB–treated TonBaF.1-NPM-ALK cells at time points indicated (representative results of 3 independent experiments). (B) Knockdown experiments using small interfering (si) RNA in the human NPM-ALK–positive ALCL cell line Karpas-299. (Top panel left) Immunoblot demonstrating changes in protein levels upon transfection with scrambled siRNA (siControl), GAPDH siRNA (siGAPDH), JUNB siRNA (siJUNB), and c-JUN siRNA (si c-JUN), respectively. (Top panel right) Cell counts (normalized to control at t = 0) demonstrate a decreased cellular proliferation of Karpas-299 transfected with siJUNB at time points indicated (representative results of 6 independent experiments. SDs for 2 biologic replicates are given). (Bottom panel) Cell-cycle analysis assessed by propidium iodide staining and flow cytometry shows a decrease in the G2/M fraction associated with an increase of the G0/G1 fraction after siJUNB transfection (representative results of 3 independent experiments). (C) EMSA of Karpas-299 cytoplasmic lysates, 24 hours after siRNA transfection with siControl, siGAPDH, siJUNB, and si c-JUN. Cell lysates were incubated with a [32P]-labeled AP-1 consensus DNA (representative results of 3 independent experiments).