The appearance of PS at the cell surface, often considered a hallmark of apoptosis, has been said to prepare the dying cell to be phagocytosed. We employed Annexin V staining to ascertain the extent of PS externalization upon treatment with the compounds. The L929 cells were treated with the IC50 for the intermediate cytotoxic compounds or with the lowest concentration tested for the highly cytotoxic compounds and were then stained with FITC-conjugated Annexin V in combination with propidium iodide and assessed by flow cytometry. Very little necrosis was induced by any of the compounds. In contrast, all the cytotoxic compounds promoted externalization of PS significantly above that induced by medium alone. Compound 5, plumbagin, served as a positive control. Caspase 3 is thought to play a central role in the execution phase of apoptosis in that it is indispensable for several hallmark features of the process. Although it has been reported that the presence of cleaved caspase 3 can be associated with non-lethal biological processes, the highly cytotoxic compound 5 and 7 have been shown to act through caspase 3. The ability of the cytotoxic compounds to promote activation of caspase 3 in treated L929 cells was therefore also assessed. All of the cytotoxic compounds activated caspase 3. The data is presented in Figure 4B, using compounds 2, 6, and 9 as representative compounds for the three cytotoxicity categories, and compound 5 as a control, Interestingly, the low/ intermediate cytotoxic compounds activated caspase 3 equivalently to the highly toxic compounds and to that of the compound 5 positive control. This Monastrol suggests that although caspase 3 is activated, the extent of activation was not proportional to the extent of cytotoxicity. To determine if the cytotoxic compounds were promoting cell death through actions on mitochondrial membrane VRT752271 potential, the DePsipher Kit from Trevigen was used. DePsipher is a unique cationic dye that indicates the loss of the mitochondrial potential. The dye readily enters cells and fluoresces bright red in its multimeric form within healthy mitochondria.