increased perinuclear staining in C6wt cells after induction of cell death by camptothecin (Fig. 6A). The observed blebbing of the nuclear membrane is consistent with the breakdown of the nuclear lamina during apoptosis. No staining for cleaved lamin protein was observed in non-stressed C6wt, non-stressed C6ko or camptothe- cin-stressed C6ko cells (Fig. 6A). Using nuclear DAPI staining as a reference point, we quantified the perinuclear immunofluores- cence in a ring around the nucleus (Fig. 6A, insets), and found a 2fold increase in staining intensity in camptothecin-stressed versus non-stressed wt MEFs (Fig. 6B). Only background signal was observed in C6ko MEFs even after camptothecin treatment, indicating that the detection of cleaved lamin A by immunoflu- orescence is also specific to caspase-6 (Fig. 6B). This method is ideally suited for the high-throughput intracellular testing of modulators of caspase-6 activity in in the presence of confounding proteolytic activity, i.e. that of caspase-3. Discussion The study of the role of caspases during apoptosis, but also during non-apoptotic developmental and signalling processes, is hampered by the lack of specific assays to measure the activity of single members of the caspase family. Neo-epitope antibodies against the active forms of caspases are available and can be used in immunohistochemical and Western blotting applications. However, the presence of active caspase fragments does not necessarily correlate with proteolytic activity, since the proteases could be bound to either endogenous or exogenous inhibitors, which is especially problematic in high-throughput screening campaigns aiming to identify caspase inhibitors. Another method using immunoprecipitation with biotinylated zVAD, on the other hand, is specific to active caspases, but quantitation relies on the efficacy of immunoprecipitation and Western blotting [23]. To be able to accurately quantify the activity of caspase-6 in cell culture, we therefore developed novel activity assays based on the cleavage of the caspase-6 specific substrate protein lamin A [24]. Here, we confirm the specificity of lamin A cleavage by caspase-6 using C6wt and C6ko MEFs that were stressed with staurosporine to undergo apoptosis. Although the antibodies we used in this study cross-react with cleaved lamin C, specificity for caspase-6 cleavage is maintained, since both proteins arise from the same gene by alternative splicing, contain the same caspase-6 cleavage site and only differ in their C-termini with lamin A being 10 kDa longer (Fig. 2D) [28,33]. The cleaved fragment was detected in C6wt, but not in C6ko MEFs, and we developed an electro- chemiluminescence-based ELISA method to accurately quantify cleaved lamin using the Mesoscale platform. The new method has an improved detection limit and signal-noise ratio over commer- cially available caspase-6 activity assays using the VEID peptide substrate. Furthermore, our assay is specific to caspase-6 and thus superior for the detection of caspase-6 activity in complex samples with a variety of different proteolytic activities such as cell lysates. Through spiking with purified lamin A protein, the assay can be applied to samples lacking endogenous lamin A and C such as embryonic primary neurons. Furthermore, the method is amena- ble to samples where caspase-6 activity does not lead to the cleavage of the endogenous nuclear lamins and is therefore not necessarily associated with apoptosis. The specificity is due to the use of a protein instead of a peptide substrate, and even though the VEID sequence is derived from the caspase-6 cleavage site in lamin A (aa227–230), we show that the Kcat/Km value for lamin A is more than 10fold higher than for VEID, indicating that lamin A is a better substrate for caspase-6. A similar effect has been shown for proteases involved in blood Caspase-6 Activity Assessment in Cell Culture Figure 6. Caspase-6 activity can be quantified by immunoflu- orescent staining for cleaved lamin A. A: C6wt and C6ko MEFs were stressed with camptothecin, stained with an antibody against cleaved lamin A and analyzed on an automated imaging platform. Nuclei were counterstained with DAPI, and identified by the software (blue circles). Debris (red circles) was not analyzed. The signal intensity in the cleaved lamin A channel was quantified in a ring around the nucleus (green circles). B: Quantitation of the perinuclear staining from cleaved lamin A is graphed. Untreated C6wt MEFs were normalized to 100% and the fold change in stressed C6wt, stressed and non-stressed C6ko MEFs are compared. Error bars are the SEM of N$3 of 3 independent experiments. Statistical significance was assessed by 2- way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001. doi:10.1371/journal.pone.0027680.g006 PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27680