The autophagy inhibitors, 3-methyladenine and Spautin-1, increase the pro-apoptotic effects of 3PO. HCT-116 cells were treated with either vehicle or 7.5 μM or 10 μM 3PO ± 1 mM 3-methyladenine (3MA). Forty eight hours after treatment, cells were stained with annexin-V and PI and measured using flow cytometry (A). Cell death was quantitated as the percentage of cells staining positive for annexin-V and PI relative to control ± SD from three experiments (B). HCT-116 cells then were treated with either vehicle, 7.5 or 10 μM 3PO ± 15 μM Spautin-1 and, 48 hours after treatment, cells were stained with annexin-V and PI and collected by flow cytometry (C). The percentage of cells stained positive for both annexin-V and PI was quantitated and is expressed relative to control ± SD from three experiments (D) (*P <0.05).