Figure 4.

Effect of Calebin A on multicellular proinflammatory TME-induced activation and nuclear translocation of p65-NF-κB in CRC cells. (A) Schematic representing the experiment model of the HCT116 cells in TME for immunofluorescence assay. (B) Serum-starved HCT116 cells in monolayer cultures alone (co) or co-cultured with fibroblasts and T-lymphocytes or co-cultured with fibroblasts and TNF-β (10 ng/ml) were either left untreated, or treated with various concentrations of Calebin A (CA) (1, 2, and 5 µM), as described in Materials and Methods. Magnification 600×; scale bar = 30 mm. All experiments were performed at least in triplicate, and quantification of positively labeled p65-NF-κB-nuclei and apoptotic nuclei (white arrows) were performed by counting 400–500 cells from 20 different microscopic fields (C). Values were compared with the control, and *p < 0.05 and **p < 0.01 were considered statistically significant.