ASK1 activation is involved in denbinobin-induced apoptosis in A549 cells. (A) A549 cells were transiently transfected with pcDNA (mock) or ASK1DN for 6 h. Following transfection, cells were replaced with fresh culture medium for 24 h and treated with vehicle or 20 μM denbinobin for another 24 h. The percentage of apoptotic cells was then determined using flow cytometric analysis of PI-stained cells as described in "Materials and methods". Each column represents the mean ± S.E.M. of at least three independent experiments. *p < 0.05, compared to the mock transfection group in the presence of denbinobin. Cells were treated with 20 μM denbinobin (B) or 5 mM H2O2 (C) for the indicated time intervals. Cell lysates were then immunoprecipitated with the anti-ASK1 antibody, and the kinase activity was measured as described in "Materials and methods". Equal loading in each lane is reflected by similar intensities of α-tubulin. 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 without denbinobin or H2O2 treatment.