Shikonin induces ROS dependent mitochondrial apoptosis in prostate cancer cells. (A) Shikonin modulated mitochondrial membrane potential in DU-145 and PC-3 cells. The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 mitochondrial dye. Briefly DU-145 and PC-3 cells were treated with 2.5 μM for 0–24 h. Ratio of 590/530 nm was measured by flow cytometry as described in material and methods. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment alters expression of mitochondrial proteins in DU-145 and PC-3 cells. Cells were treated with Shikonin for 0-24 h, expression of indicated mitochondrial proteins were performed as described in Methods. Experiments were repeated minimum two-four times. (C) Shikonin induced ROS dependent mitochondrial apoptosis in DU-145 and PC-3. Cells were pretreatment with the antioxidants NAC (20 μM) GSH (10 mM) and Catalase (20 nM) and western bolting experiments were performed as described in Methods section. Data is represents the results of three independent experiments.