Figure 5

Down-regulation of survivin with co-incubation of BPR0L075 enhances the activity of caspase-3 in KB- L30 , but not KB cells. (A and B) KB and KB-L30 cells were seeded on 8-well chamber slides overnight. Cells were pre-transfected with siR-C (scramble control) or siR-S (survivin specific) for 24 h and co-incubated with various concentrations of BPR0L075. MagicRed™-DEVD Real-time Caspase-3/-7 Activity kit (Immunochemistry Technologies LLC) was used. Activated-caspase-3/-7 was stained red and nucleus was counter-stained blue by Hoechst 33342. White arrows indicate cells with nucleus degradation. (C) Quantitative measurement of caspase-3/-7 activity. KB cells were seeded on 96-well plate and transfected with siR-C (scramble control) or siR-S (survivin specific) for 24 h. siRNA-treated cells were incubated with 4 nM of BPR0L075 for various times. Caspase-3/-7 activity was analyzed by the use of MagicRed™-DEVD real-time caspase-3/-7 activity kit with a 96-well plate-reader. (D) Analysis of DNA fragmentation by TUNEL assay. KB cells were pre-transfected with siR-C (scramble control) or siR-S (survivin specific) for 24 h and co-incubated with 4 nM of BPR0L075 for 48 h. DNA fragmentations were analyzed using the In Situ Cell Death Detection kit. Nucleus with DNA fragmentation was stained red.