Figure 5

Potential mecahnisms for cCAF-induced FN accumulation. (A) Analysis of plasminogen activator (PA) activity. Using a synthetic chromogenic substrate (Boc-Val-Gly-Arg-β NA.AcOH) there was an increase in fluorescence detected in supernatant from Rous sarcoma virus transformed fibroblasts which secrete large amounts of PA but not with the control or cCAF-treated supernatants. (B) Casein zymography to detect MMP-3, MMP-7, and MMP-10. No detectable caseinolytic activity in the supernatant of control or cCAF-treated fibroblast was found. The general protease trypsin was used as a positive control. Because this enzyme is not pure, multiple sites of protein digestion were seen. (C) Blocking TN expression using antisense oligonucleotides. Immunoblot analysis of TN and of FN; equal amounts of protein, as determined by using the DC protein assay kit (Bio-Rad),were loaded in each lane. Use of antisense TN oligos blocked the cCAF-induced expression of both FN and TN, whereas the use of sense oligos had no effect on TN or FN levels.