Figure 1

NSC23766 blocks Rac activity and RA-FLS proliferation. (A) RA-FLS were serum starved (control) in the presence or absence of 50 µM NSC23766 (NSC) for 24 h and subsequently stimulated with 10 ng/mL IL-1β for 5 min in the presence or absence of 50 µM NSC23766. Activated Rac (GTP-Rac) was extracted from cell lysates using GST-PAK and visualized by Western blotting using anti-Rac antibody. Total Rac from cell lysates before extraction was determined as loading control. Data shown are representative of two independent experiments. (B) RA-FLS (RA2) were grown overnight in 10 % serum in the absence (closed squares) or presence of 25 µM NSC23766 (open circles) or 50 µM NSC23766 (open squares). Subsequently, cells were plated on 96 well plates and cell growth was quantified using SRB staining. (C) Effect of NSC23766 on the proliferative activity of different RA-FLS cultures. Conditions as in (B). Solid bars: controls, empty bars: 50 µM NSC23766. Shown is the mean (± SEM) of five wells. For some of the data points, the error is smaller than the symbol size. * = P < 0.02, ** = P < 0.01 and *** = P < 0.001, two-tailed t test. Data for RA1 and RA2 are representative of respectively of three and two experiments. (D) Effect of NSC23766 on RA-FLS survival. RA-FLS (RA2) cells were treated with the indicated concentration of NSC23766 and apoptosis was measured using an ELISA assay that quantifies histone-associated DNA fragments. Shown are the mean values (± SEM) of three independent experiments from three RA-FLS cell lines.