Signals were detected using a chemiluminescent detection system (ECL AdvanceTM Western Blotting Detection Kit; GE Health- care) and Chemidoc XSR device (Bio-Rad). Flow cytometric assessment of apoptosis after dual staining with Annexin-V-PE and 7AAD Apoptosis was induced by a large scale of chemotherapeutic drugs in the two cell lines (MCF-7 and HBL100) (Table S2). Assessment of apoptosis was accomplished by measuring Annexin- V-PE and 7AAD staining (Apoptosis Detection Kit I BD PharmingenTM, Franklin Lakes (NJ), USA) according to manu- facturer’s instructions. Cells (30 000) were counted by flow cytometry using Becton Dickinson LSRII and the experiments were performed in triplicate in two different clones. Immunoprecipitation assays (IP) IP assays were carried out on total protein extract of HBL100 cell line with Exacta Cruz matrix (Tebu-bio, Le Perray En Yvelines, France) according to manufacturer’s instructions and analyzed by western blot. Fluorescence microscopy Wild-type, non tagged procaspase-3 or caspase-3s stably transfected MCF-7 cells were transiently transfected with procas- pase-3-GFP or caspase-3s-YFP. Cells were plated onto 6-well plates for 24 hours, then treated or not with anticancer drugs for indicated times before replacing the culture medium with phenol red-free medium. Cells were scanned in phase contrast, and with a GFP or YFP filter under UV light. Statistical analysis Data were analyzed using the student t test on the Statview 5.0 software. Results Caspase-3s inhibits drug-induced apoptosis in breast cancer cells We previously showed that in breast carcinoma, increase in caspase-3s/caspase3 ratio expression was significantly associated with chemoresistance to cyclophosphamide-based neoadjuvant treatment [6]. To go further, procaspase-3 and caspase-3s were stably transfected, either alone or in combination in MCF-7 cells. The constructs were fused to GFP (pro-caspase-3) or YFP (caspase- 3s) and the GFP vector was used as a control (Figure S1). Transfected MCF-7 cells were exposed to the topoisomerase II inhibitor etoposide, the tubulin poison vinblastine and the folate metabolism inhibitor methotrexate for 48 hours before analyzing apoptosis induction by the use of a flow cytometry assay. Expression of procaspase-3 cDNA strongly increased the ability of MCF-7 to undergo apoptosis in response to etoposide and methotrexate but did not demonstrate any significant effect on vinblastine-induced cell death in the tested conditions or slightly enhanced their apoptotic response to some of the drugs used (Figure 1A, light grey bars). Expression of the caspase-3s either did not sensitize the cells to drug-induced apoptosis (Figure 1A, black bars). Remarkably, co-expression of both procaspase-3 and caspase-3s completely suppressed the ability of procaspase-3 expression to sensitize MCF-7 cells to etoposide and methotrex- ate-induced cell death (Figure 1A, dark grey bars). These results were further completed by studying the consequences of caspase- 3s and/or procaspase-3 expression on apoptosis induced by a series of other cytotoxic agents in MCF-7 and HBL100 cells. Both Caspase-3s Inhibits Apoptosome Assembly Figure 1. Caspase-3s inhibits drug-induced apoptosis. A. MCF-7 cells were stably transfected with GFP vector (white), procaspase-3-GFP (light grey), caspase-3s-YFP (black) or with C3-GFP and C3s-YFP (dark grey) and treated with etoposide, vinblastine or methotrexate for 48 H. B. HBL100 WT cells (white) were transfected either with scRNA (grey) or with siRNA against procaspase-3 (black). The efficiency of procaspase-3 extinction is shown in the small inset. Apoptosis was next induced by etoposide, vinblastine or methotrexate and detected by flow cytometry. Asterisks correspond to p,0.01. C. HBL100 and MDA-MB-231 cells (white) were transfected either with scRNA (white) or with siRNA against caspase-3s (black). The efficiency of caspase-3s extinction is shown in the small inset. Apoptosis was next induced by etoposide, vinblastine or methotrexate and detected by flow cytometry. The drug used yields in panel C were 10 fold inferior than the IC50 used in panel B, explaining the differences between controls of panels B and C. Asterisks correspond to p,0.01. doi:10.1371/journal.pone.0029058.g001 cell lines demonstrated specific profiles of sensitivity to drug- induced apoptosis. While procaspase-3 expression (in MCF-7) or overexpression (in HBL100) increased their apoptotic response to cytotoxic agents (5FU, Bleomycin, Cisplatine, Epirubicin, Vin- cristine or Staurosporine), expression of caspase-3s always counteracted this sensitization (Table S3). Moreover, siRNA experiments targeting a sequence located in the caspase-3 specific exon 6 or a specific sequence of caspase-3s, to down-regulate the expression of procaspase-3 or caspase-3s respectively, in HBL100 cells were performed to determine the effect of endogenous PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29058