Figure 3

JSI-124 mediated c-Jun activation was not dependent on STAT3 expression in B-cell leukemia. A and B, Cells were transfected with siRNA-STAT or siRNA-c-Jun or siRNA non-target control. Then cells were treated with JSI-124 for additional 6 h. Cell extract was assayed for Western blotting for STAT3 and c-Jun. Knock-down of STAT3 or c-Jun was confirmed by western blotting with antibody to STAT3 or c-Jun. C, The same transfected cells were treated with JSI-124 for an additional 24 hours and then cells were analyzed for cell cycle analysis for G1 and G2 phase fractions in the cells transfected with siRNA-c-Jun or non-targeting control siRNA. Standard error was determined on the basis of three independent experiments and an asterisk represents significant difference (a p value of < 0.05) between JSI-124 treated and untreated controls. D, Cells were stimulated with JSI-124 and nuclear extracts incubated with the AP1 probe. Pulled down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labeled scrambled probe was used along with no probe beads control. Blots were probed with antibody to c-Jun.