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. 2019 Jun 4;9:8262. doi: 10.1038/s41598-019-44720-3

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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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Ash2l is essential and its loss interferes with histone H3 lysine 4 methylation. (a) Scheme of the wild type (wt) and floxed exon 4, and the recombined Ash2l locus. The primers used to analyze the different alleles are indicated. Drawing not to scale. The experimental design is shown at the bottom. (b) Effect of pIC treatment on Ash2l KO (n = 8) and control (n = 2) mice. Kaplan-Meier plot from the experimental endpoint. (c) PCR analysis of LSK DNA (mice treated for 10 d) using primers P3/P4 (panel a). LSK control, pool of six wt animals; LSK KO, pool of six Ash2l^fl/fl: Mx1-Cre KO animals; heterozygote, tail DNA of an Ash2l^wt/fl: CAG-CreER⁺ animal; control, no template. Floxed and recombined Ash2l alleles with 734 and 174 bp, respectively. (d) Sections of fixed sterna from control and Ash2l KO animals stained with H&E. Circles, megakaryocytes; Arrow head, granulocyte; *sinuses. (e) Total number of cells determined from 24 control and 40 KO HPF sternal sections of 4 animals each. (f) Diameters were measured of 100 chloroacetate esterase positive cells in sternal sections of 2 control and 2 KO animals. (g) H&E stainings of sternal sections containing areas of trabecular bone are displayed. Arrow heads indicate osteoblasts. (h) The length of the lining of enclosed fields of BM was measured and the number of osteoblasts determined (six areas from two animals each). (i) Total number of cells recovered from BM (2 tibias and 2 femurs/mouse) and from spleen (n = 3 each). (j) Serial tissue sections from fixed sterna stained with anti-Ash2l or anti-H3K4me3 antibodies as indicated. (k) Western blot analysis of proteins of BM cell lysates (from control (C) and Ash2l KO mice). Upper and lower halfs of the blot was probed for Ash2l and actin, respectively. (l) Flow cytometry analysis of fixed BM cells double-stained for histone H3 and H3K4me3, H3K4me1 or H3K9ac as indicated. Forward and side scatter plots (FSC and SSC, respectively) define the main population of control (blue gate) and Ash2l KO cells (red gate). These were assessed for histone H3 and the indicated histone marks. (****p < 0.0001; ***p < 0.001; **p < 0.01).