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. 2018 Dec 22;18:1283. doi: 10.1186/s12885-018-5204-x

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 1 — Confirmation of SEMA5A knockdown in PC cells and resulting mesenchymal morphology. a. Bar graph showing relative fold decrease in mRNA expression of SEMA5A. HPRT is used as a control by RT-PCR analysis. The values are mean relative fold changes ± Standard Error of Mean (SEM, bars). b-c. Western blot analysis of cell lysates of T3M-4-Control and T3M-4-shSEMA5A cells (b) and Control and CD18/HPAF-shSEMA5A cells (c) showing decreased SEMA5A protein expression with GAPDH as a loading control. Quantification of the protein of interest by the intensity of the bands with respect to their loading control was performed by Image J software. Bands were normalized on the T3M-4-Control cells or CD18/HPAF-Control cells. d-e Morphological changes upon SEMA5A knockdown. Cobblestone-like T3M-4-Control cells change to spindle-shaped cells in T3M-4-shSEMA5A cells. Furthermore, on expressing Flag-tagged mouse sema5A in T3M-4-shSEMA5A cells, the cells reverted back to their cobblestone appearance. Scale bar represents 100 μm in length (d). The transition of overlapping colonies of CD18/HPAF-Control cells to flat spindle-shape cells in CD18/HPAF-shSEMA5A cells. Scale bar represents 10 μm in length (e)