JSI-124-induced apoptosis was not mediated by c-Jun activation in B-cell malignancies. A. BJAB, I-83, and NALM-6 cells were pre-treated with 20 μM SP600125 followed by 1 μM JSI-124. Decreased levels of phospho-and total c-Jun protein levels were detected by western blotting in the cells treated with the combination of SP600125 and JSI-124 compared to SP600125 alone. Representative original data from three independent experiments are shown. The XIAP and STAT3proteins level, as determined by immunoblotting with antibodies against XIAP and serine phosphor-STAT3/STAT3, did not change with this treatment B. Apoptotic cell population was measured using flow cytometric analysis for AnnexinV/PI staining for apoptosis after 24 hour treatment with JSI-124, either alone or in combination with SP600125. DMSO-treated cells were taken as the control. Cells that were 7-AAD-negative and Annexin V-positive were undergoing apoptosis. The percentage of cells in each quadrant is indicated in the quadrant. Representative original data are shown. C. Cells were treated with same as above and were analyzed for cell cycle by FACS as described in Materials and Methods. Experiments were done at least three times and representative original data are shown (* represents significant difference of p value of < 0.05 between JSI-124 treated and untreated cells and ** represents a p value of < 0.01).