Figure 4

JSI-124 induced VEGF via activation of c-Jun. A. Increased levels of VEGF protein were detected by western blotting and mRNA by real time PCR in I-83, BJAB, NALM-6 cells after treatment with JSI-124 for the indicated times. Representative original data from four independent experiments are shown. B. BJAB, I-83 and NALM-6 cells were pretreated with 20 μmole/ml SP600125 for 1 hour following JSI-124 treatment for additional 4 hours. VEGF mRNA and protein levels were detected by RT-PCR and western blotting, respectively. 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 alone or pretreated with SP600125. C. in BJAB and I-83 cells c-Jun was knocked down by using siRNA for c-Jun or non-target control siRNA. Total RNA was extracted and real time-PCR was used to detect mRNA level of VEGF. Error bars represent the standard errors for three independent experiments. Cell extract was assayed for western blotting for VEGF. Knock-down of c-Jun was confirmed by western blotting with antibody to c-Jun. D. NALM-6 cells were treated with 20 ng/ml VEGF-165 for 1 h and followed by JSI-124 for 24 h. The percentages of apoptotic cells were determined by FACS analysis for sub-G1 accumulation.