caspase-3s after apoptosis induction. Cells were treated with etoposide, vinblastine and methotrexate for 48 hours. In absence of procaspase-3 (Figure 1B), HBL100 cells were less sensitive to apoptosis induced by etoposide and methotrexate. As expected, in absence of caspase-3s (Figure 1C), both HBL100 and MDA-MB- 231 cells were more sensitive to etoposide and methotrexate induced apoptosis, confirming that endogenous caspase-3s plays an important role in caspase-3 dependant apoptosis regulation. Caspase-3s prevents procaspase-3 activation We first analyzed procaspase-3 activation in treated MCF-7 cells transfected with pro-caspase-3-GFP or with caspase-3s-YFP. Whereas vinblastine did not demonstrate any effect on procaspase- 3 activation (Figure 2A, fifth and sixth tracks), exposure to etoposide and methotrexate induced the proteolytic cleavage of the procaspase-3-GFP into a p40 fragment indicating the activation of caspase-3-GFP (Figure 2A, third and seventh tracks). The expression of caspase-3s-YFP with caspase-3-GFP prevented the ability of etoposide and methotrexate to trigger the proteolytic cleavage of the proenzyme (Figure 2A, fourth and eighth tracks). Caspase-3s Inhibits Apoptosome Assembly In procaspase-3 proficient cells, etoposide and methotrexate induced the cleavage of caspase-3 specific substrates, the 70 KDa Lamin-A into a 28 KDa fragment and the cleavage of the 250 kDa a-Fodrin into a 160 kDa fragment [25,26]. These cleavages were not observed in MCF-7 cells expressing only caspase-3s and in those expressing both procaspase-3 and caspase- 3s in response to etoposide (Figure 2B) and methotrexate (Figure 2D). Vinblastine treatment had not effect on Lamin-A and a-Fodrin cleavage (Figure 2C) whatever the pro-caspase-3 or caspase-3s expression. Caspase-3s influences procaspase-3 location Fluorescent microscopy analyses of MCF-7 cells indicated that, in the absence of any treatment, procaspase-3-GFP was located mainly in the cytoplasm (Figure 3A, first line), whereas caspase-3s-YFP was in both nucleus and cytoplasm (Figure 3B, first line). Upon exposure to etoposide or methotrexate for 10 hours, part of the GFP-tagged caspase-3 was translocated into the nucleus (Figure 3A, second and fourth lines). After vinblastine exposure, GFP-tagged procaspase-3 remained in the cytoplasm (Figure 3A, third line). Caspase-3s Figure 2. Caspase-3s prevents procaspase-3 activation. A. MCF-7 cells stably transfected with caspase-3 were treated with etoposide, vinblastine or methotrexate (lanes 1, 3, 5 and 7). Caspase-3 was cleaved in procaspase-3 transfected cells after 48 hour treatment with etoposide, or methotrexate but not after vinblastine treatment. In procaspase-3-GFP/C3s-YFP co-transfected treated MCF-7 cells (lanes 2, 4, 6, and 8), procaspase-3 cleavage was completely abolished. b-actin was used as loading control. B–D. Analysis of lamin-A and a-fodrin cleavage by Western Blot. GFP MCF-7 cells, procaspase-3-GFP (ProC3-GFP) or caspase-3s-YFP (C3s-YFP) transfected cells were treated with etoposide (B), vinblastine (C) or Methotrexate (D) during 48 hours. Lamin-A and a-fodrin were cleaved only in single procaspase-3-GFP transfected cells treated with etoposide or Methotrexate. ProC3- GFP/C3s-YFP co-transfected cells treated with etoposide or methotrexate did not show cleavage of neither lamin-A nor a-fodrin. In accordance with panel A, vinblastine did not induce cleavage of neither lamin-A nor a-fodrin b-actin was used as loading control. doi:10.1371/journal.pone.0029058.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29058