seeded into cell culture flasks. At passage 2, cells were immortalized by transfection with pSV3-neo SV40 large T antigen (ATCC) with the Fugene reagent (Roche) according to manufacturer’s instructions. Immortalized cells were selected and propagated through the addition of 600 mg/ml G418 to the medium. For the activation of caspase-6, cells were stressed with 50 nM staurosporine for 0–6 h and harvested by trypsinization. Cell pellets were lysed in 50 mM Tris pH 8, 150 mM NaCl and 1% Igepal with 4.2 mM Pefabloc and ‘Complete’ protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation. Neuronal culture and lysis Cortical neuronal cultures were prepared as described previ- ously [38] from E16.5 littermate embryos obtained from timed- pregnant heterozygous breedings and tissues not used for culture were genotyped. Cultures were maintained at 37uC under 5% CO2 and half of the culture media was exchanged every 4–5 days. At day 10 in vitro, 5 mM camptothecin was added to the media to induce caspase activation [39], and cells were harvested after 30 h of treatment by scraping in ice-cold PBS supplemented with protease inhibitors (4.2 mM Pefabloc and ‘Complete’ protease inhibitor cocktail (Roche)). Cells were pelleted by centrifugation and stored at –80uC until lysis. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and ‘Complete’ protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation. Culture, transfection and lysis of COS7 cells COS7 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and 2 mM L- Glutamine. Cells were transfected with human caspase-6 cDNA with a C-terminal DDK tag using the Fugene reagent (Roche) according to manufacturer’s instructions. Cells were harvested 0– 24 h after transfection by trypsinization. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and ‘Complete’ protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation. Culture and lysis of MCF-7 cells MCF-7 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and 2 mM L- Glutamine. Cells were stressed with 5 uM camptothecin for different amounts of time and harvested by trypsinization. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and ‘Complete’ protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation. Western blotting 20 ng of pure lamin A protein was subjected to digestion by different concentrations of caspase-3, -6, and -7 (0.75–6 mM) in caspase cleavage buffer for 30 min at 37uC. The reactions were stopped by shock-freezing, and thawed samples were run on 4– 12% Bis-Tris gels (Nupage, Invitrogen), transferred to PVDF membranes by electroblotting and membranes were developed with primary antibodies in 5% BSA/PBS. Fluorescently labelled secondary antibodies and the LiCor Odyssey Infrared Imaging system were used for detection. Caspase-6 Activity Assessment in Cell Culture For Western blotting of cell lysates, 50 mg total protein were run on 4–12% Bis-Tris gels (Nupage, Invitrogen) and processed as described above. Cell fixation and immunofluorescent staining C6wt and C6ko MEFs were seeded at 2500 cells per well into 96well plates. N = 3 for each treatment. The following day, cells were treated with camptothecin (5 mM final) for 16 hours. Post stress, cells were washed in PBS, fixed in 4% formaldehyde (EMS)/PBS for 60 minutes at 4C, washed with PBS, and permeabilized with 0.3% Triton-X 100/PBS. Primary antibody to mouse cleaved lamin A (Cell Signaling Technology) was added at 1:100, in normal goat serum, and incubated at 4C overnight. Plates were washed twice in PBS before adding secondary antibodies, AlexaFluor 488 anti-mouse IgG (Invitrogen), each at 1:800 in normal goat serum, and DNA staining dye, Hoechst 33342 (Invitrogen), at 1:10000, for 90 minutes at room temper- ature. Plates were washed again twice and left in PBS. Image capture and immunofluorescence data analysis Labeled cells were analysed using the ThermoFisher Cellomics ArrayScan VTI, a high content scanning (vHCS) microscope, using version 6.6.2.0 software. The ArrayScan VTI captures images using an ORCA-ER camera in two channels applicable to the assay. XF-53 filters for 405 and 488 nm were used in automated image analysis to quantify nuclei number, and cleaved lamin A, respectively. The Cellomics toolbox, Compartmental Analysis, used an algorithm to encircle perinuclear staining of cleaved lamin A. The primary object in the Hoechst channel was identified as the nucleus of the cell of which 500 total were counted per well. In channels 2, the perinucleus was identified as a ‘‘circ’’, which was 3 pixels outside the primary object, where the algorithm measured pixel intensity above background staining. The ‘‘MEAN_AvgCirIntensity’’ for each channel was used for analysis, under the vHCS analysis tool, and exported to Microsoft Excel spreadsheets and GraphPad Prism for further analysis. Supporting Information Figure S1 Active site titrations for caspases -3, -6, 7. The exact active site concentration of each caspase used was determined by titrating the enzymes against the pan-caspase inhibitor zVAD-fmk [27]. (TIF) Figure S2 Km determination for lamin A. A: The indicated concentrations of lamin A protein were digested with 20 nM caspase-6 and samples were analysed with the Mesoscale ELISA system. B: Cleavage rates were determined as the slope of the curves in (A) and plotted against the lamin A concentration to obtain values for Km and kcat through curve fitting using the built-in function of the GraphPad Prism 5.0 software package. (TIF) Acknowledgments The authors thank Dr Eric Zhao for his initial work on optimizing the Mesoscale ELISA readout and Lili Liu for help with primary cell cultures. Author Contributions Conceived and designed the experiments: DEE NHS JS SL MRH. Performed the experiments: DEE NHS JS YTNN SL L-PC. Analyzed the data: DEE NHS JS YTNN SL. Contributed reagents/materials/analysis tools: DEE JS L-PC ED. Wrote the paper: DEE MRH. PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27680