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. 2017 Jun 15;7:3614. doi: 10.1038/s41598-017-03972-7

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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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Effects of consecutive transient miR-200 depletion in EAC cell lines. (A) Constitutive miR-200b/c depletion in EAC cell lines. Ishikawa and MFE-280 cell lines were transiently transfected with miR-200b/c inhibitor every 4 days for a total of 40 days (D = day, C = transfection cycle). Bar graphs represent relative miR-200b/c expression. Data are normalized to scramble. (B) Relative mRNA expression of EMT markers. TaqMan probes for ZEB1, ZEB2, E-cadherin (E-cad), N-cadherin (N-cad) and vimentin (vim) are shown on the bottom. Scramble (Scr) represents control for miR200b/c KD (200KD) samples. Data are normalized to scramble. Representative time points D12C3, D38C8 and D40C10 are shown. (C) Protein expression of EMT markers by immunoblotting. Representative early and late time points are shown. Anti-β-actin serves as a loading control. (D) Relative adhesion, and (E) migration and invasion in miR-200b/c knockdown (miR200b/c KD) vs. scramble-treated cells. (F) Relative proliferation measured by cell counts. Data are normalized to Day 0. All graphical data are reported as mean ± SD. *P < 0.05; paired sample t-test. There were no statistically significant differences in migration, invasion or proliferation between miR-200b/c knockdown and scramble-treated cells.