Figure 6: NF-κB-signalling in stem-like human prostate TICs.

Western blots of whole cell extracts of the parent tumour, spheres and the sphere tumour analyzing (a) various signalling pathway components, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) representing loading control. (b) Nuclear localization of NF-κB in stem-like sphere-forming cells. Immunofluorescence (scale bar, 100 μm) or immunohistochemistry (scale bar, 50 μm) of acetylated (K310) NF-κB visualized in the nucleus and counterstained by DAPI (IF) or hematoxylin (IHC). (c) Comparative immuno-histochemical analysis of MCL-1 expression in primary spheres versus the parent tumour, sphere tumour and human patient primary tumour. Scale bar, 100 μm. (d) Dose-dependent effects of small molecule inhibitors on secondary sphere formation. Inhibitors were administered to primary sphere-forming total tumour cells. Red, PHA (PHA 665752); brown, parthenolide; black, 481407; green, celastrol. (e) Preliminary screening to test effect of inhibitors of different signalling pathways on secondary sphere-formation in vitro. PHA (10ìμM), parthenolide (10ìM), 481407(5ìM), celastrol (2ìM), BAY (BAY11-7082, 10ìM), staurosporine (0.05ìM), MG132 (0.5ìM), LY (LY294002, 10ìM) + rapamycin (20nM), cyclopamine (2ìM) or DAPT (10ìM) were administered, wherein the control represents dimethylsulphoxide (DMSO)-treated set. (f) Primary sphere-forming tumour cells were treated with the inhibitors of MET (PHA: 20 μM), activated NF-κB signalling, (parthenolide: 20 μM; 481407: 20 μM; celastrol: 3 μM), PI3 kinase signalling (LY 294002: 15 μM)+mTOR signalling (rapamycin: 40nM) or Notch signalling (DAPT: 20 μM) were xeno-transplanted subcutaneously. Tumour size was determined by tumour volume at the 5-week end point. (g, h) Effects of these signalling inhibitors on secondary sphere formation (g) and tumour-initiation (h) across multiple human prostate tumour xenograft models. Same number of total tumour cells from human prostate DU-145 SC-(blue)/ PC-82 OT-(red) tumours or prospectively purified TRA-1-60-positive cells from the CWR22 OT-(green)/DU-145 SC-(pink) tumours were used. Tumour size was determined as above. (i) Immunoblot analyses of cleared caspase (Cl-caspase) and PARP cleavage in the primary spheres following administration of NF-κB and AKT/mTOR signalling inhibitors. Whole cell extracts prepared 6 h-post drug administration were analysed by western blotting. Control represents DMSO-treated set, whereas the total AKT and total S6RP levels served as loading controls. Mean±s.d., n≥4.