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. 2018 Feb 2;8:2268. doi: 10.1038/s41598-018-20765-8

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© The Author(s) 2018

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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The expression level of EMT induction markers. (A) Western blot analysis showing the expression level of S18-2 in PC3, PC3-S18-2-CL03 and PC3-S18-2-CL04. The graph shows the intensity of S18-2 bands, normalized to the intensity of corresponding actin bands. (B) Western blotting showed that E-cadherin and cytokeratin 8 was decreased at the protein levels in PC3-S18-2-CL04 compared with PC3 cells. The expression of β-catenin was not changed among the three cell lines. Actin and Tubulin were used as loading controls, respectively. Scans of all gels are presented in Supplementary Figure S2. (C) The q-PCR analysis of TWIST2. The TWIST2 was expressed at significantly higher levels in PC3-S18-2-CL04 than in the control cells. (D) The TWIST2 mRNA expression after 24 and 48 h of S18-2 downregulation. The TWIST2 gene was downregulated significantly upon S18-2 knocking down by siRNA in PC3 cells. (E) Expression level of S18-2 and CXCR4 in PC3 cells after 24 and 48 h of the treatment of PC3 with S18-2 specific siRNA. As expected, S18-2 was reduced with transfection of S18-2 specific siRNA compared to control siRNA treated cells. CXCR4 was also significantly reduced in cells transfected with S18-2 specific siRNA compared to control siRNA treated PC3 cells. (F) the mRNA expression level of S18-2 and TWIST2 after activation of CXCR4 by CXCL12 treatment. Cells were treated for 24 and 48 h. The TWIST2 gene was induced after 48 h. The S18-2 expression was not affected by CXCL12 treatment. All the experiments were repeated at least three times. Medians of three q-PCR reactions were analyzed, using the GraphPad Prism software. Unpaired t test was applied and two tailed p values for each experiment (controls −3, 24 h −3, 48 h −3 values) were determined.