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Mediators of Inflammation
Volume 2012, Article ID 456462, 13 pages
http://dx.doi.org/10.1155/2012/456462
Research Article

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

Laboratory of Host Defense, Department of Pharma-Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan

Received 29 February 2012; Revised 1 May 2012; Accepted 28 May 2012

Academic Editor: Luc Vallières

Copyright © 2012 Riyo Morimoto-Kamata et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Covalent complex formation between CG and AT or ACT: CG purified from neutrophils (1.67 pmol) was incubated with AT (90.9 pmol) or ACT (1.5 pmol) in RPMI 1640 medium containing 1% BSA for 10 min at 4°C. In addition, to measure the effect of an inhibitor on complex formation, CG was pretreated with chymostatin (661 pmol). Formation of the covalent complex in the mixture was determined by the presence of a heavier CG band on the western blot membrane.

Supplementary figure 1: Recombinant human CG is enzymatically active. Recombinant CC was prepared from RBL-2H3 cells that transfected with the vector encoding WT or S195G human CG cDNA. A: Enzymatic activity of CG in the cell lysates. The release rate of 4-nitroanilides was measured by adding 20-μL of the cell lysate to N-succinyl Ala-Ala-Pro-Phe p-nitroanilide in 0.1 M HEPES-NaOH (pH 7.5) containing 0.5 M NaCl and 10% dimethyl sulfoxide (180 μL) at 25°C. The released p-nitroanilide was detected by the absorbance at 405 nm. The results are shown as mean ± SD (n = 3). B: immunoreactivities of recombinant CG and β-actin in the lysates assayed in Supplementary fig. 1A. A 10-μL aliquot of the whole cell lysate were analyzed by western blotting using anti-CG and β-actin antibody. Lane 1, purified CG from human neutrophils (100ng); lane 2, the lysate of cells transfected with pcDNA3.1 that encodes of WT CG; lane 3, the lysate of cells transfected with pcDNA3.1 that encodes S195G CG; lane 4, the lysate of vector-transfected cells. The ratio of CG and β-actin protein contained in the lysates, which were quantified using ImageQuant TL, are indicated under the photographs. C: The enzymatic activity after 80 min was normalized using β-actin. The results are shown as mean ± SD (n = 3). When the bars are not shown, they are smaller than the size of the symbols.

Supplementary figure 2: Complex formation of CG and AT or ACT is abrogated by pre-treatment with chymostatin. Purified CG was incubated with AT or ACT for 10 min at 4°C. CG was pretreated with chymostatin to assess its inhibitory effect on complex formation. The formation of a covalent complex of CG and AT (or ACT) in the mixture was analyzed by western blotting using anti-CG antibody. CG, 26 kDa; AT, 50 kDa; ACT, 50 kDa.

Supplementary figure 3: PARs did not participate in MCF-7 cell aggregation induced by cathepsin G. MCF-7 cells were inoculated 1 × 104 cells/well in 96 well plate in RPMI 1640 medium supplemented with 5% FBS. The cells were cultured overnight and then incubated with a peptide of PAR-1 (A), PAR-2 (B) or PAR-4 (C) ligand in RPMI 1640 medium containing 1% BSA overnight. After washing, the residual cells were stained with crystal violet, and the aggregation index was calculated. The results are expressed as mean ± SD (n = 3). When the bars are not shown, they are smaller than the size of the symbols.

Supplementary figure 4: Scatchard plot of 125I-labeled CG binding to the MCF-7 cells.

  1. Supplementary Material