Figure 2

Effects of heparinoids partly results from inhibition of thrombin. MDA-MB231 cells were transfected with PAR-1 or control siRNA. (A) After 48 h, RNAs were extracted and subjected to qRT-PCR analysis to ascertain PAR-1 mRNA expression levels. siCtrl – control siRNA treatment. (B) PAR-1 depleted and control cells were incubated with glycosaminoglycans for 24 h, fixed and stained for F-actin. (C) Cell spreading induced by PAR-1 depletion and addition of glycosaminoglycans to PAR-1 depleted cells was quantified, n ≥ 50 cells per condition. (D) PAR-1 depleted and control cells were stained for paxillin, cadherin-11 and p120-catenin. Small focal adhesions are arrowed. (E). Levels of phosphomyosin light chain (ppMLC) are unchanged by PAR-1 depeletion by siRNA. Total myosin light chain (MLC) and β-tubulin are shown. (F) Quantification of PAR-1 and control siRNA treated cells that invaded type I collagen gels after 24 h. (G) Images and quantification of type I collagen degradation by control and PAR-1 depleted cells. Images at higher magnification are shown in the lower panels. Bars = 25 μm in B and D. Bar = 100 μm in G. Error bars = s.e.m. from three independent experiments. **p < 0.01, ***p < 0.001, n.s.: not significant. Significance was tested by one-way ANOVA with Tukey’s post-hoc test (C) or two-tailed paired t-test (A, F and G).