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. 2017 Sep 13;7:11501. doi: 10.1038/s41598-017-11914-6

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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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Generation of RWPE-1 cells stably overexpressing SEMA3C. SEMA3C was cloned under the control of a human Ubiquitin C promoter in a modified FUGW lentiviral vector designated FUGWBW using Gateway technology (a) (Invitrogen). Immortalized normal prostate epithelia RWPE-1 cells were transduced with virus made from either a SEMA3C overexpression construct to achieve constitutive expression (SEMA3C) or empty parental vector to serve as a control (FUGWBW). Overexpression of SEMA3C was confirmed by Western blot analysis of cell lysate where actin served as loading control (b). Phospho- and total levels of Akt, EGFR, and MAPK were also examined where actin or vinculin served as loading control. Western blot band intensity was quantitated by densitometry (b, right); ‘F’ = FUGWBW, ‘S’ = SEMA3C. Control cells showed cobblestone morphology which is characteristic of epithelia while SEMA3C-overexpressing cells showed cobblestone and spindle-like morphologies (c).