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. 2019 Dec 27;9:19950. doi: 10.1038/s41598-019-56276-3

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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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Down-regulation of fibronectin in 3D suspension culture reduces attachment of MDA-MB-231 parental and LM2 cells to HUVECs. (A,B) MDA-MB-231 parental and LM2 cells were re-cultivated for 2.5 hours on uncoated or fibronectin VI-coated culture glasses, fixed with 4% formalin and stained with phalloidin (green) and DAPI (blue) and (C,D) the areas covered with cells that were attached to 2D culture glasses were measured. Scale bar: 100 μm. (G) MDA-MB-231 parental and LM2 cells were re-plated on a HUVEC monolayer for 2 hours, after being cultured in 3D suspension condition for 48 hours. Breast cancer cells were stained with green CMFDA, and the nuclei of breast cancer cells and HUVECs were stained DAPI (blue). Scale bar: 200 μm. (E,F) The relative ratio of attached MDA-MB-231 parental and LM2 cells on HUVECs was measured, based on CMFDA. For the experiments the mixture of two kinds of FN1 siRNA were transfected to parental and LM2 cells. (means ± s.e.m; Student’s t-test).