Involvement of AP-1 activation in denbinobin-induced apoptosis in A549 cells. (A) Cells were pretreated with curcumin (1 μM) for 30 min before incubation with 20 μM denbinobin for another 24 h, and then apoptosis was detected using flow cytometry of PI-stained cells as described in "Materials and methods". Each column represents the mean ± SEM of at least three independent experiments. *p < 0.05, compared to the denbinobin group. (B) Cells were treated with 20 μM denbinobin for 10, 30, 60, and 120 min. Cells were then harvested and subjected to immunoblotting with an anti-phospho-c-Jun antibody as described in "Materials and methods". Equal loading in each lane is shown by the similar intensities of c-Jun. Compiled results are shown in the lower panel. Each column represents the mean ± S.E.M. of at least three independent experiments. * p < 0.05, compared to the control group. (C) Cells were incubated with 20 μM denbinobin for various time intervals. Following incubation, the nuclear protein fraction was prepared, and AP-1-specific DNA protein complex formation was analyzed by an EMSA as described in "Materials and methods". The antibodies of c-Jun and c-Fos were incubated prior to detecting the specificity of AP-1 binding activity. Fifty-fold concentrations of unlabeled AP-1 or NF-κB oligonucleotides (50× cold) were used for the competition experiment. Data shown are representative of three independent experiments with similar results.