Fibroblast cultures and wound healing assay

The fibroblast cell line NIH 3T3 (A gift from Dr Janet Daly University of Nottingham) was routinely maintained and to conduct the scratch assay published methods were used. Briefly, 3x10⁵ cells were seeded into 6 well plates and incubated overnight. To scratch the monolayer, a linear scratch was made to the fibroblasts from the top of the well to the bottom using a 20μl pipette tip at time 0; plates were then incubated with the indicated proteins for 24hrs. Images of scratches were obtained using an inverted light microscope set to 5 x magnification. Lecia imaging software (leicra microsystems LTB Milton Keynes UK) was used to acquire digital images. ImageJ, was used to analyse the images (version 1.49v from National Institutes of Health, USA). Scratch areas were measured and compared by a blinded operator independent to the culture treatments for each well.

To conduct the CFU assay 6 cells/well were seeded in a 6-well plate, thereafter proteins were added and plates incubated for 10 days. Plates were stained with 0.5% crystal violet and imaged as above. CFUs were determined per well by an operator blinded to treatments before data analysis. In some experiments cells were co-cultured with SB-431542 a TGF-β RI kinase inhibitor (Tocris) and TGF-β or FhTLM with inhibitor at a final concentration of 5μM. Inhibitor stocks were prepared in DMSO and vehicle controls were prepared using an appropriate comparative dilution of DMSO.