Validation of FLS culture. (a) Confluent monolayer of K/BxN (fibroblast-like synoviocytes) FLS as observed in vitro at 10× magnification. (b) mRNA expression of 18S, P4H, CD248, osteocalcin (OCN) and CD68 determined by standard RT-PCR at 35 cycles in one non-inflamed control FLS wild-type (WT), three K/BxN FLS lines (FLS 1 to 3), primary calvarial osteoblasts (OBs) and the macrophage cell line RAW 264.7 (MΦ). Expression of CD248 (blue), CD90.2 (magenta) and fibronectin (red) in non-inflamed FLS (c, e, g) and K/BxN FLS (d, f, h), determined by confocal fluorescence immunohistochemistry. (i) Invasion of FLS across matrigel-coated transwell inserts relative to untreated non-inflamed control FLS in the presence or absence of TNF-α (ng/ml). (j) ΔCt mRNA expression of cadherin-11 normalised for the housekeeping gene 18s. Data presented are from three individual WT control FLS and three K/BxN FLS after loading 25 ng of mRNA for RT-PCR. For Figure 1 c-h, results shown are representative of three separate K/BxN FLS lines. Figure 1b has been cropped, to allow presentation of multiple gels.