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. 2021 Jun 9;11:12186. doi: 10.1038/s41598-021-91721-2

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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EMT and metastatic properties of PC3 cells treated with immune sera. (A) Cell migration assays were performed to compare the wound healing capacity of immune serum-treated PC3^RANKL+ cells and untreated cells. Immune serum treatment decreased the migration capacity of PC3 cells, as evident from the increase in the wound healing gap at 48 h. Magnification, × 100; scale bar, 100 μm. Data from related graphs are displayed as the mean ± SD. (B) Transwell invasion assays were performed to compare the invasiveness of immunised serum-treated PC3^RANKL+ and untreated cells. Serum-treated PC3^RANKL+ cells showed a significant decrease in invasiveness. Magnification, × 100; scale bar, 100 μm. Data in the associated graphs are expressed as the mean ± SD. (C) Real-time quantitative polymerase chain reaction for the analysis of EMT and metastasis markers in PC3^RANKL+ cells. β-Actin was used as a loading control. Significant differences were observed at *p < 0.05, compared with the control. (D) Expression of E-cadherin, Vimentin, β-catenin, MMP-9, IL-6, c-MYC, TCF-4 and β-actin in immune serum-treated PC3^RANKL+ cells, as measured by western blotting. β-Actin was used as a loading control. Similar results were obtained in three independent experiments. (E) Western blot analysis of MAPK phosphorylation levels in serum-treated PC3^RANKL+ cells. Similar results were obtained in three independent experiments. (F) TOP/FOP luciferase reporter assays in immune serum-treated PC3^RANKL+ cells. Significant differences were observed at *p < 0.05 vs. the control. (G) Co-immunoprecipitation of β-catenin and TCF-4 from PC3^RANKL+ and immune serum-treated PC3^RANKL+ cells. Each blot was obtained under the same experimental conditions.