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. 2017 Nov 8;7:15101. doi: 10.1038/s41598-017-15415-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Purified recombinant HAI-2 proteins repressed cellular matriptase activation and prostate cancer cell motility. (A,B) Effects of purified recombinant HAI-2 proteins on matriptase in N2 (A) and PC-3 cells (B). Cells were treated with the indicated concentrations of rHAI-2 (Cat.1106-PI-010, R&D system, MN, USA, which was produced from mouse myeloma cells) and incubated for 20 hours. The conditioned media and cells lysates were collected for immunoblotting analysis using anti-HAI-2 pAb, M32 and M69 mAbs to detect the levels of rHAI-2 in the conditioned media and matriptase (total and activated levels) in cells, respectively. β-actin was used as control. (C,D) Effects of purified recombinant HAI-2 (rHAI-2) proteins on N2 (C) and PC-3 (D) cell migration and invasion. Cells were seeded at a density of 2 × 10⁵ (N2) or 5 × 10⁴ (PC-3) cells per well of Boyden chambers and treated with the indicated concentrations of rHAI-2 proteins. Boyden chambers were coated with or without Matrigel before cell seeding for cell invasion and migration assays. Migration assays were performed for 24 hours (N2) and 16 hours (PC-3) and invasion assays were carried out for 48 hours (N2) and 24 hours (PC-3). The results of cell migration and invasion were statistically calculated from three independent experiments and represented as mean ± SD. (*p < 0.05; **p < 0.01, one-way ANOVA).