Figure 1

Heparinoids promote adhesion and reduced invasive activity. MDA-MB231 cultures were treated with 20 μg/ml heparan sulfate (HS), chondroitin sulfate (CS), heparin (Hep) or untreated (Ctrl) for 24 h after which cells were fixed and assessed for (A) F-actin (red) to detect microfilament bundles and paxillin (green) to detect focal adhesions (arrows), (B) spread cell areas (n ≥ 50 per condition), (C) adherens junction (arrows) components cadherin-11 (green) and p120-catenin (red). (D) Sensitivity of microfilament organisation to 30 μM of Rho kinase inhibitor, Y-27632 added for 30 min before fixation and staining for F-actin, Bars = 25 μm in A, C, D. (E) Representative western blot of diphosphorylated (Thr18/Ser19) myosin light chain (ppMLC) and total MLC from cells treated with glycosaminoglycans or untreated. Quantitation for ppMLC and total MLC levels relative to β-tubulin is shown under the blots. Similar results were obtained in three independent experiments. (F) F-actin organization in cells transfected with cDNAs encoding wild type MLC (pEGFP-MLC), phosphomimetic MLC (pEGFP-MLC Thr18Asp/Ser19Asp) or control vector (pEGFP). Phalloidin-stained cultures of the same areas are shown in the inserts. Bar, 25 μm. (G) Cells were plated onto type I collagen gel coated transwells in the presence or absence of glycosaminoglycans. After 24 h, invading cells were fixed, stained with DAPI and counted. (H) Cells were cultured on native type I collagen coated plates in the presence or absence of glycosaminoglycans for 48 h. After this period, cells were removed by trypsin and degraded areas were detected as clear zones by Coomassie Blue staining. Images at higher magnification are shown on the lower panels. Quantification of matrix degradation images is shown. Bar = 100 μm. 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.