Figure 5.

BMS-345541 suppresses TNF-β- or TNF-α-induced activation and nuclear translocation of p65 and NF-κB-regulated gene end-products involved in proliferation, survival, invasion, and metastasis in HCT116 cells. (a) HCT116 cells were treated as described in Materials and Methods. They were labeled for p65 by immunofluorescence and counterstained with DAPI. Magnification 600×; bar = 30 µm. (b) All experiments were performed at least in triplicate and quantification of positively stained nuclei and apoptotic cells was performed by counting 800–1000 cells from 10 different microscopic fields. *P < 0.05, **P < 0.01. Number of NF-κB-/apoptotic-positive cells was significantly lower/higher in the BMS-345541+TNF groups than in the TNF groups. (c) Time- and (d) dose-dependent experiments of HCT116 cells in monolayer culture were performed as described in Materials and Methods. Immunoblotting of whole cell lysates was performed for anti-phospho-p65, anti-phospho-IκBα, anti-MMP-9, anti-cyclin D1, anti-Ki-67, anti- CXCR4, and anti-cleaved-caspase-3. The results are shown from at least three independent experiments and the housekeeping protein β-actin served as an internal loading control. Densitometric evaluation was performed for phospho-p65, MMP-9, cyclin D1, Ki-67, CXCR4 and cleaved-caspase-3. *P < 0.05, **P < 0.01.