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. 2019 Mar 11;9:4110. doi: 10.1038/s41598-019-40886-y

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© The Author(s) 2019

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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Neo1 knockdown disrupts adherens junctions and cytoskeletal integrity in Caco-2 cells. (a) Caco-2 cells treated with control or Neo1-siRNA and immunostained for NEO1 (green) and E-Cad (red) showed disruption of the zonula adherens (arrows). The image is a maximum projection image of a z-stack (1 μm). Scale bar-20 μm (b) Caco-2 cells stained for NEO1 (green), F-actin (red) and E-Cad (grey). Images at three apico-basal positions (1 μm, 4 μm and 8 μm) showing disruption of E-Cad localisation and reduction of basal F-Actin rich stress fibres (arrowheads). Scale bar-20 μm (c) Neo1 knockdown in Caco-2 cells was confirmed by Western blot and densitometric analysis. Representative blot with three biological replicates from one experiment and Neogenin blot has been stripped and reprobed for GAPDH. Full length blots for Neogenin and GAPDH are shown in Supplementary Fig. S1. No significant change in E-Cad protein levels after Neo1 knockdown. Each band represents cell lysate proteins from a biological replicate from three independent experiments and E-Cad blot has been stripped and reprobed for GAPDH. Full length blots for E-Cad and GAPDH are shown in Supplementary Fig. S2. (d) Tight junctions were not disrupted after Neo1 knockdown as can be seen with continuous ZO-1 staining (red). Scale bar-20 μm. (e) Western blot for ZO-1 in control and Neo1-siRNA treated cells. Full length blot for ZO-1 is shown in Supplementary Fig. S3.