Effect of Robo3 on RASF migration and aggressiveness. (a) Transfection of RASF cells (late passage) with an expression plasmid for Robo3 increased migration of rheumatoid arthritis synovial fibroblasts (RASF; n = 4) in the Boyden Chamber model to about 220% compared with control (* P < 0.05). (b) In vitro assays on cartilage destruction revealed significant induction of free glycosaminoglycans after treatment with Robo3-transfected RASF (late passages; n = 3) compared with mock-transfected cells (** P < 0.01). (c) Normal skin fibroblasts (n = 2) expressing Robo3 after transfection resulted in the release of significantly more free glycosaminoglycans than control transfected cells (* P < 0.05). (d) Matrix metalloproteinase (MMP) 1 (n = 3) and MMP3 (n = 4) mRNA expression was quantified by real-time (RT)-PCR, showing significant induction of MMP1 (* P < 0.05) and high induction of MMP-3 in mRobo3 transfected RASF (late passages) compared with control. (e) MMP1 (n = 3) and MMP3 (n = 4) protein expression from the supernatant released from SF in the GAG assay was quantified via ELISA, showing high induction of MMP-1 and of MMP3 in mRobo3 transfected RASF (late passages) compared to control (* P < 0.05).