Figure 3

Inhibition of NF-κB signalling sensitises PC3 but not DU145 cells to 17-AAG. (A) Bar graph comparing the NF-κB transcriptional activity in PC3 and DU145 cells. This was determined using an NF-κB-driven luciferase activity in transiently transfected PC3 and DU145 cells. Cells were co-transfected with either NF-κB luciferase reporter (2 μg) or pGL3 basic vector (2 μg), and with Renilla luciferase reporter (0.02 μg), and incubated for 72 h before collection and analysis. Transfection efficiencies were adjusted relative to Renilla readouts and luciferase activities were normalised to pGL3 values. Values are expressed as the mean±s.e.m. of six separate experiments, ^**P<0.01. (B) Bar graph comparing IL-8 expression levels in PC3 and DU145 cells. Real-time PCR analysis of IL-8 mRNA transcript levels was used for this comparison. Values are expressed as the mean±s.e.m. of five separate experiments, ^***P<0.001. (C and D) Survival curves representing cell viability in PC3 cells (C) and DU145 cells (D), as measured by MTT assay, conducted after a 72 h exposure to increasing concentrations of 17-AAG. The consequence of inhibiting NF-κB signalling on the effect of these compounds on cell viability was determined by co-administration of the NF-κB antagonist BAY-11-7082 (BAY) at a final concentration of 10 nM. Values are expressed as the mean±s.e.m. of five separate experiments, ^*P>0.05; ^**P<0.01. (E) Bar graph illustrating relative changes in CXCL8 mRNA transcript levels in PC3 cells after treatment with BAY-11-7082 for 6 h. Values are expressed as the mean±s.e.m. of five separate experiments, ^**P<0.01. (F) Bar graph illustrating the change in CXCL8 secretion detected by specific ELISA after treatment with BAY-11-7082 for 6 h. Values are expressed as the mean±s.e.m. of four separate experiments, ^*P<0.05.