Skip to main content
Figure 2 | Journal of Biomedical Science

Figure 2

From: Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

Figure 2

ROS generation mediates denbinobin-induced apoptosis in A549 cells. (A) Following pretreatment with NAC (1 mM) or GSH (100 μM) for 30 min, cells were incubated with the vehicle or 20 μM denbinobin for another 24 h. Cells then were harvested, and apoptosis was analyzed by flow cytometry as described in "Materials and methods". Each column represents the mean ± S.E.M. of at least three independent experiments performed in triplicate. * p < 0.05, compared to the group treated with denbinobin. (B) Cells were treated with 20 μM denbinobin, and DCF fluorescence was monitored by flow cytometry for up to 120 min as described in "Materials and methods". Results are plotted as the mean fluorescence intensity (MFI) ± S.E.M. of five independent experiments. * p < 0.05, compared to the control group. (C) Cells were pretreated with NAC (1 mM) or GSH (100 μM) for 30 min prior to denbinobin (20 μM) stimulation for 10 min. ROS generation was detected by H2DCFDA using flow cytometry. Data are represented as the MFI ± S.E.M. of three independent experiments. * p < 0.05, compared to the group treated with denbinobin. (D) Cells were pretreated with NAC (1 mM) or GSH (100 μM) for 30 min before treatment with 20 μM denbinobin for another 10 min. ASK1 kinase activity was then measured. 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 in the presence of denbinobin. Cells were transiently transfected with pcDNA (mock), AktDN (E) or ASK1DN (F) 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 10 min (for ASK1 kinase activity) or 6 h (for Akt phosphorylation). Cell lysates were then prepared and subjected to ASK1 kinase activity or Akt phosphorylation as described in "Materials and methods". Equal loading in each lane is demonstrated by similar intensities of α-tubulin or Akt1/2, respectively. Compiled results are shown in the lower panel. Typical traces are representative of two experiments with similar results.

Back to article page