TCN blocks TNF-α induced p65 nuclear translocation and the phosphorylation of IκBα.

(A) HepG2 cells pretreated with 2.5 µM TCN were stimulated with 25 ng/mL TNF-α for 20 min and processed for immunostaining with anti-p65 antibody. Nuclei of cells were stained with DAPI (blue) and p65 was visualized by green fluorescence. (B) HEK293T cells were transiently transfected with pNF-κB-Luc and p65 expression plasmids followed by pretreatment of 0.3 µM TCN and stimulation with 25 ng/mL TNF-α. Reporter activity was then measured. (C) HepG2 cells pretreated with TCN were collected after stimulation with 25 ng/mL TNF-α for 10 min. Cell lysates were then analyzed by western blot using antibodies against phospho-IκBα, phospho-p65 and IκBα. Experiments were done independently in triplicate and the results are reported as means and standard deviations. Statistical analysis was perform with Student’s t-test, *p<0.05.