Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation
Figure 4
CG binds to the surface of MCF-7 cells. (a) Detection of CG purified from human neutrophils in the MCF-7 cell surface fraction. MCF-7 cells were seeded in dishes containing RPMI 1640 medium supplemented with 5% FBS. The cells were cultured overnight and subsequently incubated with CG (209 nM) and chymostatin (33 M), AT (4.5 or 11.4 M), or ACT (75 or 188 nM) in serum-free RPMI 1640 medium for 90 min on ice. After washing, cell surface proteins were biotinylated and collected using avidin-conjugated agarose beads. The cell surface protein fractions were analyzed by western blot analysis using anti-CG antibody. (b) Immunohistochemical detection of CG on MCF-7 cells. MCF-7 cells that were treated with CG (209 nM) and a protease inhibitor (chymostatin, 16.5 M; ACT, 18.8 nM) were immunostained with anti-CG antibody in the absence of permeabilization. Scale bar = 20 m.
