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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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E2f3 expression may be reversibly repressed in E2f3 ^TRE/TRE mice. (a) Schematic of tTS(kid)-driven repression. Grey box- exon, blue box- TRE element, red oval- tetracycline regulated repressor, orange triangle- doxycycline. (b) Genotypes of live births from E2f3 ^TRE/TRE; β-actin-tTS crosses maintained on normal drinking water (H₂O), with doxycycline (dox), or with tetracycline (tetra). Chi-square test, ***P < 0.0001 (water), 0.85(dox), 0.04*(tetra). ns = not significant. (c) Immunoblot analysis of E2F3 protein levels in E2f3 ^TRE/TRE (−) and E2f3 ^TRE/TRE; β-actin-tTS (+) cultured mouse embryonic fibroblasts, developed and maintained either in the presence (dox) or absence (untreated) of doxycycline. Replicate samples represent independently isolated embryo cultures. GAPDH is included as a loading control. Refer to Supplementary Fig. 12 for full images. (d) Immunoblot analysis of E2F3 protein levels in adult control E2f3 ^TRE/TRE and E2f3 ^TRE/TRE; β-actin-tTS tissues isolated from mice maintained on tetracycline. Replicate samples are generated from tissues isolated from independent mice. GAPDH is included as a loading control, *non-specific band. Refer to Supplementary Fig. 13 for full images. (e) Quantitative RT-PCR analysis of E2f3 expression in E2f3 ^TRE/TRE; β-actin-tTS mice maintained on tetracycline (n = 3) or following 1 (n = 4) or 2 (n = 3) weeks withdrawal of tetracycline. Expression is normalized to HPRT and relative to the mean of the samples maintained on tetracycline. Error bars, s.e.m. One-way ANOVA with a Tukey’s multiple comparisons test, ***P < 0.001. (f) Immunoblot analysis of E2F3 protein levels in E2f3 ^TRE/TRE; β-actin-tTS mice maintained on tetracycline throughout (0), removed from tetracycline for 2 weeks (−2), or removed from tetracycline for 2 weeks followed by re-addition of tetracycline for 1 week (−2/+1) or 2 weeks (−2/+2). Replicate samples are generated from tissues isolated from independent mice. GAPDH is included as a loading control. Refer to Supplementary Fig. 14 for full images. (g) Schematic of rtTS driven repression. Grey box- exon, blue box- TRE element, red oval- reverse tetracycline trans silencer, orange triangle- doxycycline. (h) Quantitative RT-PCR analysis of E2f3 in control E2f3 ^TRE/TRE (n = 12 (spleen, liver), 11 (lung)) and E2f3 ^TRE/TRE; Rosa26 ^CAGrtTS/+ mouse tissues in the presence of 2 g/L doxycycline for seven days ( + 7, n = 5) or for seven days and a subsequent seven days post withdrawal ( + 7/−7, n = 4). Expression is normalized to HPRT and relative to the mean of the control samples. Error bars, s.e.m. One-way ANOVA with a Tukey’s multiple comparisons test, **P < 0.01, ***P < 0.001, n.s. = not significant.