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. 2017 Aug 30;7:9932. doi: 10.1038/s41598-017-09494-6

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© The Author(s) 2017

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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Repression of E2f3 in the absence of E2f1 and E2f2 is rapidly lethal in adult mice. (a) Histogram of erythrocytes, leukocytes, and platelets in E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE control (n = 8) and E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE; β-actin-tTS mice following withdrawal of 100 mg/L tetracycline for 1 (−1, n = 3), 2 (−2, n = 3(erythrocytes), n = 2(leukocytes, platelets)) or 3 (−3, n = 5(erythrocytes, platelets), n = 4(leukocytes)) weeks. Error bars, s.e.m. Student’s two-tailed t-test; control vs. -2weeks, **P = 0.007 (platelets); control vs. -3weeks, *P = 0.027 (leukocytes); control vs. -3weeks, ***P < 0.0001 (platelets). (b) Survival curve of E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE control (n = 18) and E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE; β-actin-tTS mice maintained in the presence of tetracycline (+tetra, n = 10) or following removal of tetracycline (off tetra, n = 13). Log-rank (Mantel-Cox) test; control vs. off tetra, ***P < 0.0001; +tetra vs. off tetra, ***P = 0.0006. (c) Haematoxylin and eosin (H&E) stained images and Ki67 expression in bone marrow, spleen, and thymus in E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE control and E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE; β-actin-tTS mice 3 weeks after withdrawal of tetracycline. Scale bar 100 µm. Representative images from n≥4 mice. (d) Quantification of nucleated cells per 40x field of view of the bone marrow of E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE control (n = 4) and E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE; β-actin-tTS (n = 6) mice 3 weeks after withdrawal of tetracycline. (e) Quantification of enteroendocrine (Chromogranin A positive), goblet (Alcian blue positive) and Paneth (lysozyme positive) cells per intact villus or per intact crypt. In all cases, a total of 30 crypts/villi were counted per mouse from the small intestine of E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE control (n = 10(enteroendocrine, goblet), n = 9(Paneth)) and E2f1 ^−/−; E2f2 ^−/−; E2f3 ^TRE/TRE; β-actin-tTS mice following the indicated number of weeks (-1 weeks, n = 2), (-2 weeks, n = 2(enteroendocrine), n = 3 (goblet, Paneth)), (-3 weeks, n = 5(enteroendocrine), n = 6 (goblet, Paneth)) post withdrawal of tetracycline. Mann-Whitney test, control vs. -3 weeks, **P = 0.004 (enteroendocrine), 0.04* (goblet), 0.005** (Paneth).