Enhanced migration of p21(-/-) fibroblast-like synoviocytes (FLS) to rheumatoid arthritis (RA) synovial fluid (SF) is independent of cell cycle regulation. (a) Wild-type (WT) and p21(-/-) FLS were treated identically to the chemotaxis assay in the presence and absence of mitomycin C (MMC) to determine whether the MMC conditions applied allowed for any proliferation of FLS. Cell counts are expressed as the percentage of cells plated, which was determined by counting the number of cells present after 2 h. The asterisk indicates a statistically significant difference between the groups, as determined from three identical experiments. (b) WT and p21(-/-) FLS were allowed to adhere to the bottom side of the membrane with the chamber inverted in the presence of 10 μg/ml MMC. Subsequently, cells were introduced to gradients of RA SFs diluted 1:75 to induce migration. Cell counts are expressed as fold-increase over background migration to PBS (negative control). An asterisk indicates a statistically significant difference. Combined chemotaxis data analyzed from seven separate patients in nine independent experiments demonstrates significantly more migration by the p21(-/-) FLS compared with WT FLS (p < 0.05). (c) A representative chemotaxis assay of 9 independent assays examining RA SF (1:75 dilution) from 7 separate patients randomly designated #5 to #11. bFGF, basic fibroblast growth factor.