Fig. 7

Inhibition of TNF-α-mediated NF-κB activation and its nuclear localization by complexes 6 and 12. (A) In the upper panel, the immunoblot shows the level of S536 phosphorylation in NF-κB phosphorylation after treating the MDA-MB-231 cells with indicated compounds (10 μM) for 6 h followed by activation with TNF-α. In the lower panel, the bar plots represent the densitometric quantification of S563 phosphorylation level after the indicated compound treatment. The error bar shows standard deviation from three independent experiments. (B) Representative confocal images of MDA-MB-231 cells showing TNF-α-mediated NF-κB nuclear localization (TNF-α treatment for 40 min) after treatment with the indicated complex for 2 h followed by activation with TNF-α for 40 min, as indicated in each panel. F-actin and the nucleus are stained with phalloidin (gray) and DAPI (blue), respectively. Scale bar, 10 μm. (C) Quantification of colocalization between NF-κB and DAPI by Menders correlation coefficient (MCC) at the indicated experimental condition. Scale bar = 10 μm. (Cell number = 120–160) in the cytoplasm of MDA-MB-231 cells suggesting that the complexes inhibit the phosphorylation and nuclear translocation of NF-κB. Furthermore, in agreement with the literature, the NF-κB inhibitory activity of 6 or 12 promotes mitochondrial stress, leading to its depolarization and the release of cytochrome C to induce caspase-3/7-mediated intrinsic pathway of apoptosis.