Caspase-6 Activity Assessment in Cell Culture Figure 4. Cleavage of lamin B1 in primary neuronal cultures is not caspase-6 specific, but spiking with lamin A provides a specific readout. A: Lamins B1 and B2 differ from lamins A and C in their caspase cleavage site (VEVD versus VEID, respectively). B: Lamin B1 is cleaved after camptothecin stress in primary cortical neuronal cultures derived from C6wt and C6ko mice. C: Spiking with lamin A protein results in an increased ELISA signal in lysates from camptothe- cin-stressed C6wt, but not C6ko neurons. Boxes represent the 25th–75th percentile, whiskers represent the minimum and maximum. Data from 5 (C6ko) and 7 (C6wt) separate cultures are shown. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001. doi:10.1371/journal.pone.0027680.g004 to the nucleus and cleavage of endogenous lamin A. To this end we transiently transfected COS7 cells with full-length human caspase-6, a system in which the enzyme auto-activates in the cytosol before translocating to the nucleus [32]. After transfection, the active form of caspase-6 becomes detectable by Western blotting at the 9 h timepoint (Fig. 5A). After 24 h of incubation, low levels of endogenous lamin A cleavage were observed with the ELISA method, indicating that at this time point active caspase-6 Figure 5. Spiking with lamin A allows for the detection of caspase-6 activity before the cleavage of endogenous lamins. A: COS7 cells were transiently transfected with recombinant human caspase-6 and cultured for the indicated amounts of time. The active caspase-6 fragment can be observed by Western blotting 9 h after transfection and is increasingly abundant after 24 h. B: COS7 cells were transfected as in A. The cleavage of endogenous lamin A protein can be observed with the ELISA method after 24 h, whereas spiking of lysates with lamin A protein leads to a robust detection of caspase-6 activity 9 h after transfection. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001. doi:10.1371/journal.pone.0027680.g005 is present in the nucleus (Fig. 5B). However, when cell lysates were supplemented with purified lamin A protein and endogenous, active caspase-6 was allowed to cleave the spiked protein, activity could already be detected after 9 h and increased dramatically at later time points (Fig. 5B), in agreement with the Western blot data (Fig. 5A). This indicates that the spiking method can detect caspase-6 activity before endogenous lamin is cleaved and is thus suitable to detect non-nuclear caspase-6 activity. High-throughput immunofluorescence measurements of lamin A cleavage Although our Mesoscale ELISA method shows significant advantages over commonly used, VEID-based assay systems to measure caspase-6 activity, it still requires manual cell lysis and protein quantification steps that are not easily amenable to high- throughput screening campaigns. We therefore turned to a high- content imaging platform that allows automated liquid handling as well as immunofluorescence imaging and quantification. Using a primary antibody specific for caspase-6 cleaved lamin A, we found PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27680