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. 2020 Jun 8;11:2869. doi: 10.1038/s41467-020-16496-y

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© The Author(s) 2020

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. 4 — a Whole-mount immunofluorescence of ear skin of 5 weeks old PIK3CA^H1047R;Vegfr3-CreER^T2 (left panels) mice showing upregulation of VEGFR3 and NRP2 expression but unchanged VEGFR2 levels in abnormal sprouts (arrows) in comparison to capillaries with normal appearance (arrowheads), and those in wild-type mice (right panels). 4-OHT (100 µg) was administered at P21. b Quantification of VEGFR3 staining intensity in the dermal lymphatic vasculature in the ear skin of 4-OHT-treated PIK3CA^H1047R;Vegfr3-CreER^T2 mice (n = 5), compared with littermate controls (n = 4) ± s.d. c Immunofluorescence staining of paraffin sections of ear skin showing increase in CD45⁺ immune cells around LYVE1⁺ lymphatic lesions in a PIK3CA^H1047R;Prox1-CreER^T2 mouse compared with a control (Ctrl). d Percentage of CD45⁺CD11b⁺F4/80⁺ macrophages relative to all CD45⁺ cells in the ear skin of 5 weeks old PIK3CA^H1047R;Vegfr3-CreER^T2 mice treated with the vehicle (n = 6) or 4-OHT (100 µg, PIK3CA^H1047R; n = 7) at P21 (mean ± s.d.). e Experimental plan for the induction of progressive microcystic LM and inhibition using the soluble VEGF-C trap (AAV-VEGFR3-Ig; AAV-sR3) or vehicle (PBS). f Whole-mount staining of ears from untreated PIK3CA^H1047R;Vegfr3-CreER^T2 mice (Ctrl), and mice treated with 4-OHT (100 µg) and AAV-sR3 or vehicle, and analyzed at different stages after induction. Images in red frame show immunofluorescence of ears from AAV-sR3 treated mice. g Quantification of lymphatic vessel branching in the progressive LM model. Red squares represent data from AAV-sR3 treated mice. Data represent mean (n = number of ears as indicated) ± s.d. Two-tailed unpaired Student’s t-test. Scale bars: 50 µm (a), 200 µm (c, f). Source data are provided as a Source Data file.