Figure 6.

EMCL inhibits NF-κB and its binding activity. (A) 786-0 and Caki-1 cells were treated with EMCL (20 µM) for 24 h following pretreatment with PDTC, an inhibitor of NF-κB (30 µM), for 6 h, and the cell viability was determined by Cell Counting Kit-8 analysis. (B) 786-0 was either untreated (lane 2, control cultures) or treated with the indicated doses of EMCL (lanes 3-5) for 24 h. Nuclear extracts were prepared and analyzed by an electrophoretic mobility shift assay. (C) NF-κB-dependent gene expression in the 786-0 cell line. Cells were transfected with Bwt-luc reporter plasmid, stimulated with EMCL (5, 10, and 20 µM). These stimuli were administered 24 h following transfection, and the cell lysates were prepared following an additional 24 h incubation for the determination of luciferase activity. As an internal control, pRL-TK, expressing Renilla luciferase under the control of TK promoter, was co-transfected. All luciferase activities were corrected by the internal control activity of Renilla luciferase. Data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^**P<0.01, vs. dimethyl sulfoxide-treated group. NF-κB, nuclear factor-κB; EMCL, epoxymicheliolide; PDTC, ammonium pyrrolidine dithiocarbamate; Ctrl, control.