cJun phosphorylation is mediated by the JNK group of MAPKs in NPM-ALK⁺ ALCL cells. (A) SU-DHL1 cells were treated with the specific JNK inhibitor, SP600125, at concentrations of 0, 10, 20, or 40 μM. Whole-cell lysates were prepared at 48 hours and immunoblots showed that inhibition of JNK activity resulted in decreased ^Ser73p-cJun levels in a dose-dependent manner. This decrease was detectable at a concentration of 10 μM, but it was more prominent at a concentration of 20 μM. A decrease in cJun protein level was also observed because of an autoregulatory mechanism of cJun on its own transcription. (B) Treatment of SU-DHL1 cells with U0126, a specific inhibitor of the MEK1/2 group of MAPK kinase, was performed at concentrations of 0, 10, and 20 μM. After incubation for 48 hours, whole-cell lystates were prepared and immunoblots showed decreased ERK1/2 phosphorylation, indicating sufficient ERK inactivation. However, this did not result in decreased cJun phosphorylation. These results suggest that JNKs, but not ERK, mediate cJun activation in NPM-ALK⁺ ALCL. (C) JNK activity was assessed using an in vitro kinase assay using GST-cJun as a substrate. SU-DHL1 cells were treated with the specific JNK inhibitor, SP600125, at concentrations of 0, 20, and 40 μM. After 24 hours of incubation, cells were collected and whole-cell lysates were prepared and incubated with the GST-cJun substrate. Immunoblots showed that inhibition of JNK activity is associated with decreased cJun phosphorylation in vitro. (D) SU-DHL1 cells were transfected with AP-1-Luc reporter plasmid and, after incubation for 24 hours, were treated with the specific JNK inhibitor SP600125 at concentrations of 0 and 20 μM. After 24 hours the cells were collected to determine luciferase activity. The graph shows a substantial decrease in AP-1 promoter activity after JNK inhibition. Bars indicate standard error. (E) To further investigate the role of JNK1/2 isoforms in cJun activation, SUDHL1 cells were transiently transfected with 20 μg of siRNA selectively targeting JNK1 or JNK2 gene products, and endogenous JNK1/JNK2 expression levels were confirmed by RT-PCR, as well as by Western blot analysis. Cells were harvested at 48 hours after transfection and mRNA and whole lysates were prepared. RT-PCR and immunoblots confirmed JNK1 or JNK2 silencing. cJun phosphorylation was decreased after knocking down JNK1 or JNK2 in immunoblots. (F) SU-DHL1 cells were transiently transfected with 0, 10, and 20 μg of JNK1 or JNK2 or control siRNA, and whole cell lysates were prepared at 48 hours after transfection. Immunoblots demonstrate upregulation of p21 protein levels in a concentration-dependent manner.