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. 2020 Mar 5;11:1204. doi: 10.1038/s41467-020-14956-z

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 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. 3 — a Schematic of Lewis Lung Carcinoma (LLC) tumor allograft assay in endothelial-specific Cds2 knockout mice. b Representative tumor images from Vehicle Control (Cad5(PAC)-CreERT2;Cds2^lox/lox); Cre, TMX Control (Cad5(PAC)-CreERT2 +TMX); and endothelial-specific Cds2 genetic deletion mice (Cad5(PAC)-CreERT2;Cds2^lox/lox +TMX). Bar = 1 cm. c, d Quantitation of LLC average tumor volume (c) and final tumor weight (d) at 14 days post- tumor implantation in the eight genetic conditions tested. Data in c are normalized to the starting day 4 tumor volume of Wild type + TMX controls. e Representative western blot images of EC protein isolated from tumors for each indicated genetic condition, probed for CDS2 and tubulin as a loading control. Blots demonstrate that CDS2 protein is still reduced in Cad5(PAC)-CreERT2;Cds2^lox/lox mice at day 14, after only a 5 day pulse of TMX treatment initiated prior to the start of the experiment. p ≤ 0.05, error bars ± SEM. *Significance from control (t-test). f, g Quantitation of average tumor vascular density (f) and average liver vascular density (g) of CD31/PECAM labeled LLC tumor and liver sections from control Wild type +TMX, control Cad5(PAC)-CreERT2 +TMX, control Cad5(PAC)-CreERT2;Cds2^lox/+ +TMX, and endothelial-specific Cds2 genetic deletion Cad5(PAC)-CreERT2;Cds2^lox/lox +TMX mice. For all vascular density measurement experiments: three images per tumor were acquired and vascular density measured for all groups. A minimum of two slide sections from each tumor (taken from sections at least 10 slices apart) were analyzed. p ≤ 0.05, error bars ± SEM. Star indicates significance from control (t-test). h–k Representative images of CD31/PECAM labeled blood vessels in tumors from control Wild type +TMX, control Cad5(PAC)-CreERT2 +TMX, control Cad5(PAC)-CreERT2;Cds2^lox/+ +TMX, and endothelial-specific Cds2 genetic deletion Cad5(PAC)-CreERT2;Cds2^lox/lox +TMX mice. Bar =100 μm. Naming key: Wild type + TMX (no Cre, no Cds2 lox cassette; n = 18); Cad5(PAC)-CreERT2 +TMX (Cre^iECΔ only, +TMX; n = 13); Cad5(PAC)-CreERT2 (Cre^iECΔ only, no TMX vehicle control; n = 9); Cds2^lox/lox +TMX (only homozygous Cds2 lox cassette, +TMX, no Cre; n = 6); Cds2^lox/lox (only homozygous Cds2 lox cassette, no TMX vehicle control; n = 9); Cad5(PAC)-CreERT2;Cds2^lox/+ +TMX (Cre^iECΔ, Cds2 heterozygous lox cassette, +TMX; n = 16); Cad5(PAC)-CreERT2;Cds2^lox/lox +TMX (homozygous lox cassette, Cre^iECΔ, +TMX− experimental deletion group; n = 12); Cad5(PAC)-CreERT2;Cds2^lox/lox (homozygous lox cassette, Cre^iECΔ, no TMX vehicle control; n = 7). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box, respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers.