The growth kinetics of RA FLSs at different passages or in SF-treated cultures. (a) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) at passages 3 (P#3), 5 (P#5) and 11 (P#11) were used to measure their growth kinetics. RA FLSs (2–14) at passage 11 was treated with diluted synovial fluid (SF) no. 2 (1/100, 1/50 or 1/10 dilutions) to evaluate growth recovery; the culture was incubated for 6 days and the concentrations of treated SF were as follows: left panel, 1/10 dilution; right panel, 1/100, 1/50 and 1/10 dilutions of SF. *p < 0.01. (b) The concentration of granulocyte – macrophage colony-stimulating factor (GM-CSF) and cytokines in SFs from each patient with RA. Six SF samples were analyzed for their concentrations of GM-CSF and other cytokines with an ELISA kit and a Cytometric Bead Array kit. The results in (a) and (b) are means ± SD obtained from single experiments performed in triplicate cultures. (c, d) MLN51 expression was quantified by RT-PCR (c) and Western blot analysis (d) in SF-treated or GM-CSF-treated RA FLSs. The high-passage-number RA FLSs (RA 2–14, passage 11) were treated with GM-CSF (100 ng/ml) or 1/10-diluted SF every 2 days for 6 days (left panels). In addition, the expression of MLN51 was evaluated in six different RA FLS samples treated with or without GM-CSF (100 ng/ml; right panels). See the Materials and methods section for a detailed description. Data in (c) and (d) are representative of three separate experiments.