Chloroquine increases the pro-apoptotic effects of 3PO against LLC cells and tumors in vitro and in vivo . LLC cells were treated with 25 μM 3PO for 24 hours and LC3-II levels were measured using immunoblotting (A) and quantitative densitometry (B). Levels expressed as mean fold change LC3-II/β-actin relative to control ± SD (B). LLC cells were then treated with either vehicle or 10 or 25 μM 3PO ± either 15 or 30 μM CQ. After 24 hours of treatment, cells were stained with annexin-V and PI and measured using flow cytometry (C). Cells staining positive for both annexin-V and PI were quantitated as the percentage of the total relative to control and data is presented as the mean ± SD from three experiments (E). C57/BL6 mice were inoculated with 1x106 LLC cells by subcutaneous flank injection. Mice were randomized into four treatment groups when tumors reached 150–200 mm3 and were treated by i.p. injections with either vehicle, 50 mg/kg CQ, 0.07 mg/g 3PO, or a combination of the two drugs. Tumor measurements taken over the course of treatment were used to calculate tumor mass. Data is presented as mean tumor mass ± SD (D). Tumors were fixed, paraffin embedded, and stained with an antibody directed against cleaved caspase-3 (CC3) (F). The number of cells staining positive for CC3 in five 200X fields were counted and the data is expressed as the mean ± SD from three counts (G) (*P <0.05).