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. 2017 Jun 14;7:3515. doi: 10.1038/s41598-017-03747-0

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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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Fluoxetine reduces the membrane-localized E-cadherin and increases E-cadherin endocytosis. (A) After 3-hour incubation with or without fluoxetine (30 μM), cellular proteins were harvested by using a biotin-surface protein isolation assay, and then analyzed by western blotting. E-cadherin in total lysates, membrane fractions and cytosol fraction are shown. β-actin was used as a loading control for total protein. Hsp90 (heat shock protein 90) was used as a loading control for cytosol fraction. PMCA (calcium pump pan PMCA ATPase) was used as a loading control for membrane fraction. The representative data was from at least 3 independent experiments. Cropped blots have been presented. Full length blots are presented in Supplementary Fig. S6. (B) Quantitative analysis of total E-cadherin, membrane E-cadherin and cytosolic E-cadherin expression. Values (means ± SEM) are from three independent experiments. *P < 0.05 (C) After 3-hour fluoxetine (30 μM) treatment, MIN6 cells were fixed and then stained. The representative confocal images show the localization of E-cadherin (green) and EEA1 (red) in MIN6 cells. Nuclei were stained with Hoechst 33258 (blue). White arrows indicate the co-localization of E-cadherin and EEA1. Scale bar, 5 μm. The representative images were from three independent experiments. (D) Co-localization ratio between E-cadherin and EEA1 with pixel-by-pixel analyses. Each value represents mean ± SEM from at least 30 different cells from three independent experiments. ***P < 0.001.