The cells used in these experiments were passage matched. all control and inhibition experiments were run in parallel. Floating collagen gel contraction assay Experiments were performed essentially as described previously. Briefly, 24 well tissue culture kinase inhibitor Oligomycin A plates were precoated with bovine serum albumin. Normal and SSc lesional fibroblasts were treated with TGFb or PDGF with or without ERK inhibitor U0126, the ALK5 inhibi tor SB 431542, the PDGF receptor inhibitor Gleevec, or IFNb for 24 h. Pretreated fibroblasts were suspended in MCDB medium and mixed with collagen solution, pH 8. 0, four parts collagen and five parts of MCDB 2 yielding a final concentration of 80,000 cells per ml and 1. 2 mg/ml collagen. Collagen/cell suspension was added to each well.

After polymerisation, gels were detached from wells by adding 1 ml of MCDB medium with PDGF, TGFb or tumour necrosis factor b. Contraction of the gel was quantified by loss of gel weight and decrease in gel diameter over a 24 h time period. siRNA knockdown Specific siRNA recognising TSP1 was purchased as a pool of three predesigned siRNAs alone with a recom mended control siRNA. Normal and SSc fibroblasts were transfected using Silen cer siRNA Transfection II Kit. Cells were transfected either with control siRNA or control siRNA with TSP1 siRNA. Western blot analysis with an anti TSP1 antibody was performed to check the efficiency of the siRNA to reduce TSP1 protein expression. The contractile ability of the cells was analysed as described above.

GSK-3 Results Blocking TSP1 activation of latent TGFb with LSKL peptide decreased the enhanced contractile activity of fibrotic SSc fibroblasts Both overexpression of TSP1 and elevated TGFb activity can be found in SSc dermal fibroblasts. We wanted to evaluate whether TSP1 mediates matrix con traction Diabete in fibroblasts by assessing if interfering with binding of TSP1 to TGFb suppresses the basal and TGFb induced contractile activity of normal or SSc fibroblasts. LSKL peptides and SLLK peptide were used in the FPCL assay of matrix contraction. Fibroblasts in the three dimensional FPCL system generate contractile forces, similar those found in scars and in granulation tissue undergoing matrix remodelling during normal and pathological situations. Healthy and SSc fibroblasts were pretreated with TSP1 blocking peptides or control peptide for 5 days and then transferred to a culture force monitor and forces exerted by cells within the collagen lattice over 24 h in 2% serum, both in the presence and absence of added TGFb were mea sured and recorded.