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Fig. 2. Effects of calpain antagonists on on cGMP-induced motility and calpain activity. (A) Motility of NO-activated cells with and without 10 µM of the calpain inhibitor Calpeptin. There was very little cell movement of Calpeptin-treated osteoclasts relative to cGMP-activated cells (8-pCPT-cGMP). In cGMP-activated cells with Calpeptin added (middle bar), motility was reduced relative to cGMP-activated cells by 60%, with considerable variation between cells. However, the effect relative to 8-pCPT-cGMP-only treatment is significant (*P<0.05), averaging measurements over 20 cells ± s.d. (B) Effect of Calpeptin, calpastatin and calpastatin scrambled peptide on calpain activity in osteoclasts. Calpain activity was determined using the BOC assay as described for Fig. 1, in osteoclasts without stimulus or treated with the cGMP agonist 8-pCPT-cGMP or the NO donor SNP. Note that the scrambled calpastatin gives results essentially congruent with the controls (right two versus left two bars), whereas Calpeptin and calpastatin inhibited activity to below control cells and prevented calpain activation after cGMP or NO stimulation by more than 90%. Average of four experiments ± s.d. (left two bars) and of two experiments ± s.d. (right six bars) with each experiment measuring fluorescence in 
40 cells. *P<0.05 relative to control unstimulated cells (first bar). 
P<0.01 relative to control stimulated cells (second bar). (C) Cell diameter decreased after cGMP was activated in Calpeptin-treated cells that did not show significant linear translocation. (Top panel) Group of three osteoclasts and a single smaller cell, probably an un-fused CD14 derived macrophage. Calpeptin and 8-pCPT-cGMP were added added at the time the top phase photograph was taken. (Middle and bottom panels) No linear translocation but the footprint of the cells shrank (compare encircled cell). Cell diameter decreased 15.7±5.1% at 15 minutes and 18.1±1.4% at 30 minutes (n=3, mean ± s.d.). After 30 minutes the mean diameter did not change significantly (not shown). Cell diameter also decreased with cGMP activation only (Yaroslavskiy et al., 2005), but not with Calpeptin treatment alone (not shown). Thus, NO-induced rearrangement of the cell attachment does not solely depend on calpain.
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