N-acetylcysteine prevents 3PO-induced ROS and autophagy. HCT-116 cells were treated with either vehicle or 10 μM 3PO ± 1 mM NAC and harvested after 8 hours of treatment for measurement of DCFDA fluorescence using flow cytometry (A, D). Cell lysates also were prepared and LC3-II levels were determined using immunoblotting (B) and densitometry (C). Last, HCT-116 cells were stained with 1 μg/mL acridine orange for 15 minutes and acridine orange fluorescence was determined using flow cytometry (E, F). Data are presented as the mean ± SD from three experiments (*P <0.05).