Figure 7. Shh pathway affects lung fibroblast migration, invasion and collagen synthesis.

(A) Accumulated distance of migration of primary human lung fibroblasts treated with Shh (500 ng/ml) or cyclopamine (10 µM) and monitored by live cell microscopy for 48 hours. The accumulated distance of migration of each cell was determined using ImageJ. ***p<0,01. (B) Scratch wound assay was performed in lung fibroblasts CCL206 treated or not with Shh at the doses indicated or with 10 µM cyclopamine (cyclop) for up to 48 hours. Migration of the cells was recorded using live cell microscopy and representative pictures at 1,5, 12,5 and 26 hours are shown. The colored lines indicate the border of cell migration in each case. (C) The area of the wound was quantified after 26 hours and the percentage of wound closure, relative to the initial area of scratch for each case, was determined. (D) Transmembrane invasion assay was performed in lung fibroblasts treated with Shh (500 ng/ml) or with cyclopamine (10 µM). (E) RT-qPCR was performed to assess MMP2 and MMP9 expression in fibroblasts treated or not with Shh (500 ng/ml) for 48hr. Results are presented as fold of mRNA levels in treated cells compared with non-treated cells. *p<0,1. (F) The total collagen content of lung fibroblasts treated with Shh (500 ng/ml) or TGF-ß1 (5 ng/ml) for the indicated times was quantified using the Sircol collagen assay. *p<0,1, **p<0,05.