Inhibition of cJun expression reduces cell growth in NPM-ALK⁺ ALCL cells. (A) NPM-ALK⁺ ALCL cells were transiently transfected with 10 μg or 20 μg (SU-DHL1) and 20 μg or 40 μg (Karpas 299) cJun or control siRNA, and whole-cell lysates from both cell lines were prepared at 48 hours after transfection. Western blot analysis showed that endogenous cJun was almost completely silenced when 40μg siRNA was used. As expected, decreased levels of ^Ser73p-cJun were also observed. (B) Silencing of cJun gene expression in NPM-ALK⁺ ALCL cells resulted in decreased AP-1 DNA binding activity as shown by EMSA and autoradiography (first row, black arrow). SU-DHL1 and Karpas 299 cells were transiently transfected with 20μg c-Jun siRNA. After incubation for 48 hours, nuclear extracts were prepared and assessed by EMSA using double-stranded consensus AP-1 oligonucleotide. Mutant AP-1 oligonucleotide served as a negative control (second row). Free probe (FB, DNA oligonucletides) is shown in the bottom panel. (C) Selective silencing of cJun gene expression resulted in decreased cell viability (top panel) and a more prominent decrease in proliferation of viable cells (bottom panel) as assessed by trypan blue exclusion and MTS assays, respectively. The data presented are the mean (± SD) in triplicate measurements. (D) Reduced cell proliferation in NPM-ALK⁺ ALCL cells is attributable to cell-cycle arrest, because cJun silencing also resulted in decreased S-phase fraction evaluated by BrdU incorporation studies. Results have been normalized to those of control siRNA samples. (E) Western blot analysis after transient transfection of NPM-ALK⁺ ALCL cells with specific cJun siRNA showed upregulation of p21 and underphosphorylated Rb in a concentration-dependent fashion, as well as downregulation of cyclin A and cyclin D3.