Caspase-3s Inhibits Apoptosome Assembly Figure 5. Caspase-3s interacts with procaspase-3 and inhibits the apoptosome assembly. A–B. This experiment was carried out in HBL100 cells stably transfected with caspase-3s-YFP or YFP alone and treated with etoposide, vinblastine or methotrexate for 48 hours. A. In YFP transfected cells, immunoprecipitations were performed with anti-caspase-3 antibody and western blot with anti-GFP (that recognizes YFP in YFP transfected cells and the caspase-3s YFP tag) and anti-caspase-3 antibodies. The results revealed no interaction between caspase-3 and YFP with or without treatment. In HBL100 transfected with caspase-3s-YFP, an interaction between procaspase-3 and caspase-3s after etoposide or methotrexate treatments was observed. B. In YFP transfected cells, immunoprecipitations were performed with anti-GFP antibody and western blot with anti- caspase-3 and anti-GFP antibodies. The results revealed no interaction between YFP and procaspase-3 whatever the conditions. In caspase-3s-YFP transfected HBL100, an interaction between caspase-3s and procaspase-3 was observed after etoposide or methotrexate treatment. C–F. MCF-7 cells were transfected with empty-vector (YFP), procaspase-3 (ProC3) or caspase-3s-YFP (C3s-YFP) or both caspase-3s-YFP and procaspase-3. The cells were treated or not with etoposide. C. Immunoprecipitations were performed with anti-active caspase-9 antibody and western blot with anti-active caspase-9, anti-Apaf-1, anti-caspase3 and anti-GFP (that recognizes caspase-3s tag) antibodies. The results showed that procaspase-3 and caspase-3s interacted, in treated cells, with active caspase-9 and anti-Apaf-1 only when they are alone. As soon as caspase-3s and procaspase-3 are present at the same time, the interaction with active caspase-9 and anti-Apaf-1 was abolished. D. Immunoprecipitations were performed with anti-caspase-3 antibody and western blot with anti-caspase-3, anti-Apaf-1, and anti-active caspase-9 antibodies. The results showed that procaspase-3 interacted with active caspase-9 and anti-Apaf-1 in treated cells only in absence of caspase-3s. E. Immunoprecipitations were performed with anti-GFP antibody and western blot with anti-GFP, anti-Apaf-1 and anti-active caspase-9 antibodies. The results showed that YFP did not interact with active caspase-9 and anti-Apaf-1 whereas caspase-3s interacted with active caspase-9 and anti-Apaf-1 in treated cells only in absence of procaspase-3. F. Immunoprecipitations were performed with anti-Apaf-1 antibody and western blot with anti-Apaf-1, anti-active caspase-9, anti-caspase-3 and anti- GFP antibodies. The results showed that procaspase-3 and caspase-3s interacted, in treated cells, with active Apaf-1 and caspase-9 only when they are alone. As soon as caspase-3s and procaspase-3 are present at the same time, the interaction with Apaf-1 and active caspase-9 was abrogated. doi:10.1371/journal.pone.0029058.g005 PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29058