Shikonin induces reactive oxygen species mediated ER Stress. (A) Shikonin treatment induces ER stress associated protein expression. Western blot experiments in prostate cancer cells treated with 2.5 μM of Shikonin increased the phosphorylation of PERK, and its substrate eIF2α, increased expression of GRP78/Bip and CHOP/GADD135 in a time-dependent manner. (B) DU-145 and PC-3 cells were pretreated with either NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 h and treated with Shikonin (2.5 μM) for 24 h and the indicated protein levels were measured using Western blotting. Representative data of three independent experiments are presented.