Degradation of fluorescent ECM networks by SPARC.

(A) Deposition of fluorescently labeled ECM. NIH/3T3 fibroblasts were grown on Matrigel-covered coverslips in the presence of green AF488-labeled collagen. Fluorescent collagen was incorporated into the normal 3-dimentional ECM networks, deposited by live cells. After cells were removedby mild lysis, the fluorescently labeled matrix remained intact. (B) Networks were formed by NIH3T3 cells with green AF488-labeled collagenand red AF594-labeled fibronectin. After cell removal (NO cells), fluorescent networks remained intact in tissue culture conditions for at least 48 hours. SPARC knock-out fibroblasts (KO cells) grown on pre-formed fluorescent matrix re-arrangedthe networks by bending, merging and thickening fibrils around the cells. Wild type cells (WT cells) also remodeled the fibrils and caused matrix degradation, which was almost complete in 48 h. (C) Quantitative image analysis of the above experiment with separation of red (fibronectin) and green (collagen I) colors. Measured by the loss of fluorescence, matrix degradation was statistically significant (p<0.05) starting at 2 h after the attachment of the wild type cells (WT), compared with untreated matrices (NO). SPARC knock-out fibroblasts (KO) did not cause statistically significant decreasein fluorescence (p>0.05).