Figure 3

TG2 expression and matrix degradation are increased in FLSs from CIA rats. (A) Control FLSs (C-FLSs) and arthritic FLSs (A-FLSs) were cultured for 72 hours, and TG2 in the ECM laid down by the cells was visualized with immunoblotting. Graph shows the densitometric analysis of TG2/actin ratio (n = 4) with representative immunoblot (insert). (B) C-FLSs and A-FLSs were cultured on gelatin, and TGase activity was measured by using in situ 5-(biotinamido)-pentylamine incorporation assay. Graph shows mean labeling intensities of 20 cells per experiment (n = 3). (C) Representative confocal microscopy image (60×) of the basal surface of the cell showing localization of endogenous F-actin (red), p-cortactin (blue), and TG2 (green) and overlay of the three channels (merge) in RA-FLSs grown on gelatin for 16 hours. Arrows show the position of the actin and p-cortactin dots that colocalize with TG2. (D through G) A-FLSs were transfected with FXIIIa-, TG2-, or their respective control (scrambled)-shRNA-expressing lentivirus. (D) The percentage of invadopodia-forming cells was counted for 100 GFP-expressing cells (TG2-shRNA) or 300 cells (FXIIIa-shRNA), and (E) the mean area of degradation per cell was calculated for 25 transfected cells (n = 3). (F) Representative immunoblot of TG2 or FXIIIa and actin showing knockdown with respective shRNA. (G) In situ TGase activity was measured in a minimum of 20 cells (n = 3). Values are expressed as the mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).