Inhibition of JNK activity reduces cell growth due to G2/M cell-cycle arrest in NPM-ALK⁺ ALCL cells. (A) SU-DHL1 cells were treated with increasing concentrations (0, 10, 20 μM) of SP600125. Inhibition of JNK activity resulted in decreased cell viability and proliferation of viable cells as assessed by trypan blue exclusion (left panel) and MTS assays (right panel), respectively. Data presented as the mean (± a standard deviation [SD]) in triplicate measurements. (B) The decrease in cell growth of SU-DHL1 cells, after treatment with the inhibitor SP600125 for 48 hours, was associated with cell-cycle arrest. The S-phase of the cell cycle was assessed by BrdU incorporation (left panel) and measured by a colorimetric assay. Results were expressed as percentage of cells in the S-phase of cell cycle (mean ± SD of triplicate measurements) compared with untreated cells. Cell-cycle analysis, assessed by propidium iodide staining and flow cytometry (right panel), revealed a dramatic decrease in the S-phase fraction associated with a more than 3-fold increase of the G2/M fraction after treatment of SU-DHL1 cells with 20 μM SP600125. (C) Western blot analysis after treatment of SU-DHL1 cells with SP600125 at the same time point (48 hours) showed increased levels of the cyclin-dependent kinase inhibitor p21, a known cell-cycle regulator for transition to S and through G2 phase. In addition, increased levels of underphosphorylated retinoblastoma protein and decreased levels of cyclin A were observed in a dose-dependent manner.