coagulation [34,35], where substrates are bound outside the active site of the protease (exosite). There has been speculation about the presence of exosites in caspases [36], but their existence has not been proven conclusively yet. The example of exploiting protein substrate specificity for the development of an activity assay could be used for the development of more specific assays for other members of the caspase family, such as p23 for caspase-7 [3]. Caspase-6 has recently emerged as an important player in neuronal dysfunction and degeneration and its activation has been linked to several neurodegenerative conditions such as AD, HD and stroke [16,17,18,19,20,21,22,30,37]. Direct inhibition of the enzyme as well as targeting of other players in the activation pathway might therefore be beneficial in these disorders. We show here that the cleavage of lamin A can be assessed in a high- throughput setting using a high-content imaging platform, making it an ideal intracellular readout to test interventions that decrease caspase-6 activity. Materials and Methods All experiments were carried out in accordance with protocols (Animal protocol A07-0106) approved by the UBC Committee on Animal Care and the Canadian Council on Animal Care. Ac-VEID-Afc, Ac-DEVD-Afc, Ac-VEID-CHO, zVAD-fmk and active caspase-3, -6 and -7 enzymes were purchased from Enzo Biosciences. Lamin A antibodies were from Cell Signaling Technology: cleaved lamin A and total lamin A/C for Western blotting (cat. nos. 2031 and 2032), cleaved lamin A for Mesoscale ELISA (cat. no. 2036) and immunofluorescence staining (cat. no. 2036). Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full- length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R). Camptothecin and staurospor- ine were from Sigma, cell culture reagents were from Gibco. Activity assays using Ac-VEID-AFC and Ac-DEVD-AFC The amounts of enzyme indicated in the figures (5–500 nM) were incubated with 100 mM Ac-VEID-AFC at 37uC in caspase cleavage buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT) for 1 h in a black 96well plate (Nunc). Fluorescence was measured every 5 min with excitation at 400 nm and emission at 505 nm. The initial linear part of the curve was analyzed. For the calculation of active site concentrations, Ac-DEVD- AFC was used for caspases-3 and -7 according to the same protocol, and different amounts of zVAD-fmk were added to all reactions. The ratio between reaction velocity with inhibitor (Vi) over reaction velocity without inhibitor (V0) was plotted against the inhibitor concentration, and the concentration for y = 0 was determined as the active site concentration of the caspase. The specific activity of the caspase enzymes used was determined with a standard curve using free AFC and the Ac- VEID-AFC or Ac-DEVD-AFC substrate for caspase-6 and caspases -3 and -7, respectively. The resulting specific activities were: 11.7 nmol Ac-VEID-AFC/min/mmol caspase-6, 13.7 nmol Ac-DEVD-AFC/min/mmol caspase-3 and 9.9 nmol Ac-DEVD- AFC/min/mmol caspase-7. For the assessment of VEID cleavage in cell lysates, lysates corresponding to 100 mg protein were mixed with an equal volume 200 mM Ac-VEID-AFC in 26 caspase cleavage buffer. Fluorescence was measured as described above. Caspase-6 Activity Assessment in Cell Culture Determination of Km and Kcat Different concentrations of Ac-VEID-Afc substrate (1–150 mM) were digested with 10 nM caspase-6 in caspase cleavage buffer for 1 h in a black 96well plate (Nunc). Fluorescence was measured every 5 min with excitation at 400 nm and emission at 505 nm. The initial linear part of the curve was analyzed. Different concentrations of lamin A protein (3–400 nM) were digested with 20 nM caspase-6 in caspase cleavage buffer for 15, 30, 45, and 60 minutes at 37uC. The reactions were stopped by shock-freezing, and thawed samples were analysed with the Mesoscale ELISA system. The cleavage rate was calculated at each lamin concentration and plotted against the concentration of Lamin A protein to calculate the Km and Kcat (Fig. S2). Caspase-6 activity assays using the Mesoscale ELISA system 100 ng pure lamin A protein was subjected to digestion by different concentrations of caspase enzymes as indicated in the figures (0.05–1000 nM) in caspase cleavage buffer for 30 min at 37uC. The reactions were stopped by shock-freezing, and thawed samples were analysed with the Mesoscale ELISA system: 5 ml of each reaction, corresponding to 25 ng lamin A protein, were spotted onto Mesoscale ELISA plates. Samples were incubated at room temperature for 1 h, then 150 ml 5% BSA in PBS was added per well and the plate was again incubated for 1 h at room temperature. All wells were briefly washed 3 times with 150 ml PBS containing 0.05% Tween-20, and 25 ml antibody mix was added per well (cleaved lamin A antibody, Cell Signaling 2036, 1:100, goat anti-mouse sulfo-tag secondary antibody, MSD technology, 1:500 in 1% BSA/PBS). After 1 h incubation at room temperature, all wells were briefly washed 3 times with 150 ml PBS containing 0.05% Tween-20, and 150 ml 26 reading reagent (MSD technology) was added per well. The plates were then read on a Mesoscale platform electrochemiluminescence reader (MSD technology) according to manufacturer’s instruc- tions. For assays using MEF or MCF-7 cell lysates, lysates were diluted in PBS to 0.2 mg/ml, 5 ml were spotted in each well of a Mesoscale ELISA plate and the plate was developed as described above. For assays using neuronal or transfected COS7 cell samples, lysates corresponding to 20 mg total protein were mixed with 100 ng lamin A protein in 16 caspase cleavage buffer. Samples were incubated for 3 h at 37uC, 5 ml were spotted in each well of a Mesoscale ELISA plate and the plate was developed as described above. Mouse embryonic fibroblast culture and lysis Mouse embryonic fibroblasts (MEFs) from a C6ko mouse and its C6wt littermate were generated from day 12.5 embryos resulting from timed-pregnant heterozygous breedings and tissues not used for culture were genotyped. The generation and characterization of C6ko mice is described elsewhere [27]. Single pups were dissected in ice cold PBS, the body without head, limbs and liver, lung and heart was minced, the centrifuged pellet was digested with 0.25% Trypsin-EDTA at 37uC for 15 min and neutralized with MEF medium (Dulbecco’s modified Eagle medium with high glucose, 10% fetal calf serum, 2 mM L- Glutamine, 100 mM non-essential amino acids, 1 mM sodium pyruvate, 1 mM b-mercaptoethanol). Cells were passaged through a pipette tip, centrifuged and suspended in MEF medium containing DNase I. After centrifugation, cells were suspended in fresh medium, incubated for 2 min at room temperature to let debris and cell clumps settle and cells in the supernatant were PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27680