Fig. 5

Comparison of sheddase activity in mouse embryonic fibroblasts (MEFs), NCI-H460, and primary human fibroblasts. (a) Lysates from MEFs genetically deficient in a disintegrin and metalloproteinase (ADAM10 −/−) or (b) from NCI-H460, synovial fibroblasts, lung fibroblasts, or skin fibroblasts transfected with control or ADAM10 siRNA were analyzed for cadherin-11 cleavage in the presence or absence of ionomycin (representative of at least three experiments, fibroblasts (Fb). ADAM10 siRNA silencing was confirmed by western blot and β-actin levels confirmed equal protein loading. (c) C-terminal fragment 1 (CTF1) and ADAM10 expression was measured by calculating the mean pixel intensity of CTF1 and ADAM10 bands across several experiments in control and ADAM10 siRNA treated cells for the indicated cell types (H460, n = 3; ^* P = 0.049 by paired t-test; lung and skin fibroblasts, n = 3; synovial fibroblasts, n = 6, five separate rheumatoid arthritis (RA) lines; error bars reflect standard error of mean). ADAM10 silencing efficiency (mean+/−standard deviation): H460 cells, 77.0 + 5.67%; synovial fibroblasts, 70.2+/−25.5%; lung fibroblast, 85.0+/−8.04%; skin fibroblasts, 58.8+/−7.77%. (d) Cell lysates from indicated cells treated overnight with the metalloproteinase inhibitor batimastat (10 μM) or appropriate dimethyl sulfoxide (DMSO) vehicle control were assayed for cadherin-11 cleavage in the presence and absence of ionomycin stimulation (representative of at least three experiments per cell type). Equal protein loading was confirmed by β-actin staining