Invasive potential in co-culture of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and monocytes/macrophages or THP-1 cells, and RA/osteoarthritis (OA) FLS alone. The invasive potential was evaluated in transwell chambers as described in Patients and methods. Briefly, cells were suspended in serum-free media supplemented with or without AP-9 or SSP (200 μg/ml) and seeded into the upper side of the matrigel (5 μg/ml)-coated chambers. After incubation for 24 hours at 37°C, the number of invaded cells was determined using a colorimetric H & E assay. Black arrow, FLS; white arrow, THP-1 cells (f × 200). (a) and (d) The invasive potential of RA FLS was higher than that of OA FLS (P < 0.05; six samples with RA FLS, four samples with OA FLS). (b) and (d) the invasive potential of RA FLS co-cultured with undifferentiated THP-1 cells (uTHP-1) and differentiated THP-1 cells (dTHP-1) and the invasive potential of co-cultured cells inhibited by AP-9 (P < 0.05, RA FLS+ uTHP-1 vs RA FLS+ uTHP-1+ AP-9 and RA FLS+ dTHP-1 vs RA FLS+ dTHP-1+ AP-9; n = 6, respectively), while no inhibitory effects were found in irrelevant peptide SSP (P > 0.05, RA FLS+ uTHP-1 vs RA FLS+ uTHP-1+ SSP and RA FLS+ dTHP-1 vs RA FLS+ dTHP-1+ SSP; n = 6, respectively). (c) The invasive potential of RA FLS co-cultured with monocytes/macrophages from synovial fluid of RA patients (Mo-SF) and monocytes from peripheral blood of healthy humans (Mo-PB) and the invasive potential of co-cultured cells inhibited by AP-9 (n = 10, P < 0.05), while no inhibitory effects were found in irrelevant peptide SSP (n = 10, P > 0.05). Values are the means ± standard error (n = 4–10). *indicates P < 0.05 (experimental groups versus control groups). Stars indicate P < 0.05 (RA FLS co-cultured with dTHP-1 versus RA FLS co-cultured with uTHP-1, and RA FLS co-cultured with Mo-SF versus RA FLS co-cultured with Mo-PB).