Fig 3.

XDH/XO inhibition blocks Alternol-induced ROS accumulation and apoptosis. A-C; PC-3 cells were seeded in 24-well plates overnight and were then loaded with total ROS fluorescent probe for 30 min. Cells were treated with Alternol (10 μM) or Febuxostat (20 μM) as indicated for 4 h and cell nuclear were stained with Hoechst 33342 (1.0 μg/ml) for 15 min before assessment for ROS-positive cell population under a fluorescent microscope. Microscopic images were taken from each treatment and the representative images were shown in panel A. Quantitative data were summarized in panel B as described [22]. Cell death rate was determined after 8 h following drug treatment by trypan blue exclusion assay as shown in panel C. A statistical significance was indicated by the asterisk compared to the solvent control or by the hashtag compared to the Alternol alone (Student t-test, p < 0.05).

D-E; PC-3 cells with or without XDH siRNA (100 nM) transfection for 2 days were treated with Alternol (10 μM) or Febuxostat (20 μM) for 12h as indicated. A scramble siRNA was used as negative control in panel E. Cells were harvested in cold PBS and protein lysates were used for Western blots with the primary antibodies as indicated on the left side of the panel. Actin blots served as protein loading control. Data represent two separate experiments.