Caspase-3s Inhibits Apoptosome Assembly apoptosome assembly. For that, MCF-7 cells were transfected with a vector encoding either YFP alone or the YFP-tagged caspase-3s or procaspase-3 without any tag. These cells were left untreated or treated with etoposide for 48 hours. Afterwards, cell lysates were immunoprecipitated with an anti-active caspase-9 antibody and Apaf-1 antibody representing the Apaf-1/cyto- chrome c/caspase-9 complex, precursor of apoptosome. The active form of caspase-9 was detected by immunoblot analysis in etoposide treated cells (Figure 5C, Active caspase-9 panel) and its interaction with Apaf-1 was confirmed in etoposide treated samples (Figure 5C, APAF-1 panel). The results showed an interaction between active caspase-9/Apaf-1 and procaspase-3 (as detected with an anti-procaspase-3 antibody) or caspase-3s (as detected with an anti-GFP antibody, recognizing caspase-3s tag) when these proteins were expressed alone in etoposide-treated cells. These interactions were no more detected when both procaspase-3 and caspase-3s were expressed simultaneously (Figure 5C, Procaspase-3 and C3s-YFP panels). The results were confirmed by inverse immunoprecipitation with an anti-procas- pase-3 antibody (Figure 5D), an anti-GFP antibody (Figure 5E), Figure 6. Model of apoptosis inhibition by caspase-3s. After apoptotic signal, cytochrome c is released from mitochondria to the cytoplasm. Then, Apaf-1/cytochrome c oligomers are formed and interact with caspase-9. In presence of procaspase-3 only, the complex Apaf-1/cytochrome c/ active caspase-9 interacts with procaspase-3 to form the complete apoptosome. Then, active caspase-3 is release from the complex and apoptosis occurs. In presence of caspase-3s, the complex Apaf-1/cytochrome c/active caspase-9 interacts with caspase-3s to form a ‘‘short’’ apoptosome having not the ability to induce apoptosis. Finally, in presence of both procaspase-3 and caspase-3s, caspase3s interacts with procaspase-3. This interaction induces the sequestration of procaspase-3, inhibiting the complete apoptosome assembly. doi:10.1371/journal.pone.0029058.g006 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29058