Shikonin treatment induces ROS generation, increase in Intracellular Calcium in prostate cancer cells. (A-B) Shikonin induces ROS generation in prostate cancer cells. DU-145 (A) and PC-3 (B) cells were seeded in 6-well plates overnight in the presence or absence of Shikonin, and incubated with DCF-DA 50 μM for 30 min, washed three time with ice cold PBS. Generation of ROS was measured using flourimetry as per standard protocol. (C) Shikonin induces increase in intracellular Calcium in Shikonin treated prostate cancer cells. The level of intracellular calcium in DU-145 and PC-3 cells were determined using flourimetry technique in cells using the Fluo-4AM (50 μM/ml) dye in Shikonin treated prostate cancer cells as described in material and methods. (D) Shikonin treatment induces calpain activity in prostate cancer cells. Shikonin treated prostate cancer cells were analyzed for calpain activity as described in as described in material and methods. (E-F) Antioxidant pretreatment reverses shikonin induced increase in the intracellular calcium. DU-145 and PC-3 cells were pretreated with NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 hours and the ROS inhibitor rescued intracellular calcium release was measured as described in the Methods section. Values are represented as Mean ± SEM, from three independent experiments.