Figure 4

TGase activity of TG2 is involved in in vitro matrix degradation by FLSs. (A) C-FLSs were incubated with TGF-β (20 ng/ml), DTT (1 mM), or cystamine (100 μM ). After 3 hours, TG2 expression was evaluated with Western blotting (n = 2), and TGase activity was measured for cells incubated on gelatin matrix (n = 7). (B) C-FLSs or C-FLSs transfected with TG2-shRNA-expressing lentivirus or (C) A-FLSs were cultured on a gelatin matrix, incubated with TGF-β (20 ng/ml), DTT (1 mM), cystamine (10 or 100 μM), KCC-009 (25 or 250 μM), or Z-DON (1 or 100 μM), and the percentage of cells forming invadopodia at 24 hours was counted for 300 cells per experiment (n = 3). (D, E) C-FLSs were transfected with wild-type TG2 (TG2 WT), TG2-W241A, or empty vector and cultured on gelatin for 24 hours. (D) The percentage of cells forming invadopodia for 100 transfected cells (overexpressing TG2) per experiment (n = 3) and (E) the mean area of degradation per cell calculated for 25 transfected cells (n = 3) is shown. (F) Human C-FLSs treated with TGF-β (5 and 10 ng/ml) or DTT (0.1 and 1 mM) or (G) synoviocytes from patients with rheumatoid arthritis (RA-FLSs) transfected with GFP-tagged control- or TG2-shRNA-expressing lentivirus or treated with cystamine (10 and 100 μM) were grown on gelatin, and the percentage of GFP-expressing invadopodia-forming cells was counted at 24 hours (n = 3). Values are expressed as the mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).