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. 2019 Jun 24;10:2761. doi: 10.1038/s41467-019-10707-x

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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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Fig. 7 — Ccm3^−/− cells recruit and induce endothelial-to-mesenchymal transition in Ccm3^+/+ cells. a, b Lung immortalised Ccm3^−/− endothelial cells expressing LifeAct-mCherry and Ccm3^+/+ cells expressing LifeAct-EGFP were co-cultured for 7 days, with spheroids formed, showing experimental scheme (a) and representative confocal images (b). c Non-labelled Ccm3^+/+ and LifeAct-mCherry expressing Ccm3^−/− cells were co-cultured for 7 days, with the spheroids formed detached by shaking the culture plate. The detached spheroids were collected and added to an already formed monolayer of LifeAct-EGFP Ccm3^+/+ cells (see also Supplementary Fig. 7 for more details). d Representative confocal images of the basal and more apical levels of spheroids where Ccm3^−/− (red) have recruited Ccm3^+/+ cells (green). e Representative three-dimensional reconstructions of spheroids with Ccm3^+/+ cells (green) recruited. Scale bars 50 μm (b, d). f Quantification of migratory rates of Ccm3^+/+ endothelial cells assessed using the classical wound-healing assay. After the scratch, cells were incubated with medium conditioned by either Ccm3^+/+ cells (negative control) or Ccm3^−/− cells and without or with the BMP inhibitor DMH1. Data are means ± SE. p < 0.001 among groups (one-way analysis of variance (ANOVA)); *p < 0.01 (Tukey’s post hoc test); n = 3 independent experiments. g Labelled Ccm3^+/+ and Ccm3^−/− cells co-cultured as described above. After 7 days, spheroids were carefully detached, disaggregated and the cells separated by fluorescence-activated cell sorting (FACS). The cells that remained at the bottom were trypsinised and Ccm3^+/+ cells were separated by FACS. In parallel, Ccm3^−/− cells were cultured separately as positive control and processed together with spheroids. After FACS, gene expression was analysed by RT-qPCR. Data are means ± SE; p < 0.01 among groups (one-way ANOVA); *p < 0.01 (Tukey’s post hoc test); n = 4 independent experiments. h Ccm3^+/+ endothelial cells were incubated for 7 days with medium conditioned by either Ccm3^−/− cells or Ccm3^+/+ cells (negative control), and the gene expression was analysed by RT-qPCR. Data are means ± SE. *p < 0.01 (Student’s t test); n = 3 independent experiments. Source data are provided as a Source Data file