Currently, the role of caspase-3s in chemotherapy response is unexplored. We also previously studied the impact of caspase-3s expression in a population of breast carcinomas treated with neoadjuvant cyclophosphamide-based chemotherapy and ob- served an inverse relationship between caspase-3s/caspase-3 ratio level expression and pathological response [6]. Therefore, the present study was performed to precise the molecular mechanism in apoptosis inhibition of caspase-3s by using breast tumor cell lines MCF-7 treated by various chemotherapeutic agents known to induce an apoptotic mode of cell death. Materials and Methods Cell lines Human breast cancer cell lines MCF-7 (deficient for caspase-3), HBL-100 and MDA-MB-231 (proficient for caspase-3) were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA). The cell lines were cultured according to the manufacturer’s instructions. Full-length cDNA synthesis and cloning The full-length of caspase-3 and caspase-3s coding sequences were obtained using SuperScriptTM One-Step long templates RT- PCR (Invitrogen, Carlsbad, CA, USA) with 1.25 mg of total RNA from the UACC3199 cell line (Arizona Cancer Center Tissue Culture Shared Resource) containing high levels of the two transcripts. Specific primers used were reported in Table S1. PCR program was performed by one cycle at 45uC for 30 min, 94uC for 2 min followed by 35 cycles of 15 s at 94uC, 30 s at 50uC, 1 min at 68uC, and one final cycle for 5 min at 72uC (Abi Prism 9700 thermocycler, Applied Biosystems, Foster City, CA, USA). The two transcripts were separated by a 3% agarose gel electrophoresis and purified by specific extraction with QIAquick Gel Extraction Kit (Qiagen, Courtaboeuf, France). The inserts were cloned into pcDNA3.1/CT-GFP-TOPO or pcDNA3.1/CT-YFP-TOPO and amplified in One ShotH TOP10 Chemically Competent E. Coli with Fusion TOPOH TA Expression Kits (Invitrogen). The plasmids from a few randomly picked colonies were isolated. The orientations of the caspase-3 or caspase-3s fragments were tested by PCR and automatic sequencing as described below. Stable transfection For stable transfection, 56105 MCF-7 and HBL100 cells were grown in a medium without antibiotics in 12-well plates. Two days later, cells were transfected with 0.5 mg of plasmid containing either caspase-3 or caspase-3s insert and with GFP or YFP control vector. Stable transfections were performed by LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. The stable colonies were selected by 1000 mg/ml GeneticinH or 10 mg/ ml Blasticidin (Invitrogen). To ensure that the inserts were sufficiently expressed, the transfection efficiency was controlled by quantitative real-time RT-PCR amplification, the presence of GFP and YFP was controlled by microscopy and the presence of extrinsic caspase-3 and caspase-3s was determined by Western Blot. Relative change expressions were calculated between control and transfected cells. Caspase-3 and caspase-3s extinction by siRNA HBL100 or MDA-MB-231 cells (36104 per wells), which are proficient for caspase-3, were grown in a medium without antibiotics in 6-well plates during 2 days. Transfections (procas- pase-3 siRNA: CGACUUCUUGUAUGCAUACUCCACA, cas- pase-3s siRNA: GGGTTATTATTCTTGGCGAA) were per- Caspase-3s Inhibits Apoptosome Assembly formed by LipofectAMINE 2000. Twenty four, 48, 72 and 96 hours of extinction were tested by Western Blot (Figure S2A) or quantitative PCR (Figure S2B). Cytotoxic treatments were performed during the best extinction time lapse (Figure S2A and B). For treatment after caspase-3s down-regulation, chemothera- peutic drug yields were 10 fold inferior to the IC50. Sequencing of PCR products The specificity of all PCR amplifications was verified by sequencing of PCR-products. Briefly, products were excised from 3% agarose gels and isolated as described above. The purified PCR products were sequenced using the Abi-Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with the respective primers used in the initial PCR according to the manufacturer’s protocol. RNA extraction, cDNA synthesis, and Quantitative real-time PCR amplification Total RNA was extracted with Trizol reagent (Invitrogen) and its quality was checked by 28S/18S ratio on agarose gel. One microgram of total RNA was reverse transcribed as previously described [6]. For real-time PCR, amplification was performed in a total volume of 25 mL in the presence of 600 nM of each primers, 200 nM of probe, 12.5 mL of Universal Master Mix (Applied Biosystems), and 12 ng of cDNA (or water as negative control). PCR was performed with an initial denaturation step of 10 min at 95uC, followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. All samples were amplified in duplicate and results were analyzed at the CT level. Control 18S reactions (Applied Biosystems) were used to normalize DCT values. Total protein extractions Total protein extraction was performed by addition of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM b-Glycerophosphate, 1 mM Na3VO4, 1 mg/ml Leupeptin, 1 mM PMSF added immediately prior to use) on cell monolayer for 5 min on ice. Afterwards, the lysate was harvested and sonicated. Western Blot analysis 30 mg of protein extracts were separated SDS-PAGE 10% acrylamide and transferred to PVDF or nitrocellulose membranes (according to the molecular weight of the protein) with an electrophoretic transfer apparatus. After blocking with buffer containing 3% of ECL AdvanceTM blocking agent (GE Health- care, Buckinghamshire UK) in PBS/Tween 0.3% for 1.5 hour at room temperature, the membrane was probed with specific polyclonal antibodies (Abcam, Cambridge, UK) diluted in blocking solution (b-actin 1/25000, caspase-3 1/7500, GFP 1/ 50000, caspase-9 1/1000, APAF-1 1/1000, lamin A 1/4000, or a- fodrin 1/2000) for 1 hour at room temperature. The antibody used for procaspase-3 and caspase-3 detection was generated with the epitope residues between amino acids 161 and 175. This part of the protein is common to procaspase-3 and caspase-3 as it is located in the large subunit. The GFP antibody recognizes YFP in YFP transfected cells and thus the caspase-3s YFP tag. Next, the membrane was incubated with biotinylated anti-rabbit IgG (1:50000 in blocking solution) secondary antibody (Invitrogen) 30 min at room temperature. Then, the membrane was probed with streptavidin-HRP (1/ 50000) for 15 min at room temperature in PBS/Tween 0.3%. PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29058