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. 2019 Oct 30;9:15645. doi: 10.1038/s41598-019-52218-1

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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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Lef1 regulates Wnt5a clustering and the expression of caveolin family members. (a) Knock-down of Lef1 reduces the formation of Wnt5a clusters at the plasma membrane of DMZ cells. Both dorsal blastomeres of Xenopus 4-cell stage embryos were injected with 150 pg Wnt5aeGFP mRNA together with the membrane marker GAP43mCherry and 1 pmol of the Lef1 specific antisense morpholinos (LefMo) or 500 pg dominant negative Lef1 mRNA (Lef∆HMG). The arrows indicate some Wnt5a clusters located at the plasma membrane, arrowheads point to cytoplasmically located Wnt5a clusters. (b) Downregulation of Lef1 reduces the presence of Wnt5a clusters at the plasma membrane. Shown are the relative membrane localization according to Manders et al.⁵³ and the p values according to Student’s t-test. The images were processed using the Coloc 2 plugin in ImageJ which allows calculation of the Manders coefficient M2⁵³. This coefficient indicates colocalization of Wnt5aeGFP clusters and the red coloured membrane. (c) In transfected HEK293 cells, Lef1∆HMG and wild-type Lef1 do not significantly regulate the non-canonical Wnt reporter ATF2Luc. (d) In Wnt5a stimulated cells (cotransfected with Wnt5aeGFP), Lef1∆HMG represses ATF2Luc activity. Wild-type Lef1, instead, activates the ATF2Luc reporter. p values of at least nine independent transfections were determined according to Student’s t-test. e) Real-time RT-PCR confirmed that the expression of cavin1, caveolin1 and caveolin3 are downregulated in Lef1 morphants and partially restored in morphants co-injected with 500 pg Lef1 mRNA. Shown are mean values and standard error of four biological replicates.