Figure 3.

Acacetin stimulates the JNK/c-Jun pathway by inducing ROS generation in OS cells. (A) Cells were treated with increased concentrations of acacetin for 24 h, followed by loading with 10 μM DCFH–DA for 30 min. The level of ROS was determined by fluorescence microscopy. (B) Fluorescence intensity was detected using flow cytometry. Cells were preincubated with GSH (5 mM) for 2 h and then treated with acacetin for 24 h. Fluorescent intensity was detected by flow cytometry. Results are presented as mean±S.D. from three independent experiments. (C) Cells were treated with various concentrations of acacetin for 24 h. The expression of p-JNK, JNK, p-c-Jun, Jun and Cyt-c was analyzed by Western blotting. (D and E) SJSA and HOS were preincubated with SP600125 (30 μM) or GSH (5 mM) for 2 h and then treated with acacetin for 24 h. Levels of p-JNK, JNK, p-c-Jun, Jun and Cyt-c were analyzed by Western blotting. *P<0.05 and **P<0.001, significantly different compared with the control.