Proteasome activity is increased by TNFα stimulation and lysosome inhibition. (a) Validation of the chymotrypsin activity assay. Fibroblasts were cultured in EBSS for 2 hours with the addition of 0.5 μM epoximicin or 50 μM calpain inhibitor XI. The substrate Suc-Leu-Leu-Val-Tyr-AMC and digitonin were then added to the wells and fluorescence was measured every 5 minutes for a 45-minute period. (b) Fibroblasts were cultured with or without 10 ng/ml TNFα for 2 or 24 hours before substrate addition. Slopes were determined and relative proteasome activity is represented as the slope of TNFα-induced cells relative to their non-induced controls. Ctl, control; RA, rheumatoid arthritis. (c) Fibroblasts were cultured with 12.5 μM chloroquine (CQ) for 2 or 24 hours prior to addition of the substrate. Results are presented as the ratio of the activity in the presence of CQ compared with the absence of CQ. (d) Fibroblasts were cultured for 1 hour with 12.5 μM CQ before the addition of 10 ng/ml TNFα for 2 or 24 hours. Results are presented as the ratio of the activity in the presence of CQ plus TNFα compared with that in the presence of TNFα. Significant differences in TNFα-stimulated compared with non-induced cultures of control cells or significant differences between the response of control cultures compared with the response of RA synovial fibroblast cultures: *P < 0.05, **P < 0.01.