Chloroquine increases the number of late apoptotic cells caused by exposure to 3PO or PFKFB3 siRNA transfection. Annexin-V/PI staining was performed in cells treated with vehicle, 3PO, CQ, or the combination of 3PO and CQ for 48 hours. Quantitation of cell staining was performed using flow cytometry (A) and the number of cells staining with both annexin-V and PI was quantitated and is expressed as the percentage relative to control ± SD from three experiments (B). HCT-116 cells also were transfected with either a negative control siRNA or with a PFKFB3-specific siRNA ± 30 μM CQ. After 48 hours of transfection, cells were stained with annexin-V and PI and measured using flow cytometry (C). Quantitation of cells staining positive for annexin-V and PI was performed relative to control and is expressed as the mean ± SD from three experiments (D) (*P <0.05).