MDA-MB231 cell collagen gel invasion requires MMP14 and is slowed by antithrombin III treatment. (A) Various concentrations of antithrombin III (ATIII) were used to treat MDA-MB231 cells for 24 h followed by fixation and staining for F-actin. The representative images shown were cells treated with 3 μg/ml of ATIII. Bar = 25 μm. (B) Quantification of spread cell areas in response to ATIII treatment, n ≥ 50 cells per condition. (C-F) Cell invasion after 24 h (C, E) or degradation (D, F) of type I collagen gels after 48 h in the presence or absence of 3 μg/ml antithrombin III (C, D) or 50 μM MMP inhibitor GM6001 (E, F). Higher magnification images are shown in the lower panels of D and F. Bars =100 μm. (G) Cell lysates were prepared 48 h after transfection with siRNA targeting MMP14 or control siRNA. Western blotting confirmed the siRNA-mediated MMP14 depletion in the treated cells. (H, I) MMP14 depleted cells were subjected to type I collagen cell invasion and degradation assays. Higher magnification images are shown in the lower panel (I). (J) Relative cell surface expression of MMP14 in PAR-1 depleted cells was assessed by flow cytometry. Error bars = s.e.m. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.; not significant by two-tailed paired t-test.