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. 2018 Sep 25;8:14335. doi: 10.1038/s41598-018-32729-z

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© The Author(s) 2018

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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Eed deletion impairs TEC proliferation, survival and differentiation. (a) Percentage of fetal and newborn Eed^CKO versus control TEC that incorporate BrdU after injection into pregnant or newborn mice. (b) qRT-PCR analysis of Cdkn2a expression in E17.5 TEC isolated by FACS sorting. Expression in Eed^CKO TEC is normalized to control TEC. Each sample was analyzed in triplicate and the experiment was repeated twice. (c) Percentage of cleaved caspase 3 (CC3) positive Foxn1+ TEC in P0 Eed^CKO and control thymi. The frequency of apoptotic TEC was determined by counting the total number of Foxn1+ TEC that were positive for CC3 on each of 3 different sections/thymus. Results shown are from three independent experiments. (d) Frozen sections from P0 thymi were stained for K8 (green), K5 (red) and K14 (blue). White arrows indicate K8+K5+K14− TEC near the CMJ; this subset is widely distributed in controls, but sparse in mutant thymi. Note also the smaller medullary regions identified by K8−K5+K14+ TEC in Eed^CKO thymi. White bar indicates scale (250 μm). (e) Representative FACS plots of EpCAM+ CD45− TEC from P0 mice showing the distribution of UEA+Ly51− mTEC and UEA− Ly51+ cTEC subsets in control and Eed^CKO thymi. (f) Representative FACS plots showing expansion of the MHCII^lo TEC subset in P0 Eed^CKO thymi. (g) Bar graph showing percentage (mean ± SD) of cTEC and mTEC subsets in control and Eed^CKO thymi. (h) Bar graph showing number (mean ± SD) of cTEC and mTEC in control and Eed^CKO thymi. Significance was calculated using an unpaired t test corrected for multiple comparisons using the Holm-Sidak method (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).