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. 2019 May 8;10:2110. doi: 10.1038/s41467-019-10100-8

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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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Fig. 2 — Characteristic features of the EMT-associated ribosome biogenesis program. Cell culture experiments in b–k depict proliferating (Control) and TGFβ-treated (TGFβ) NMuMG cells at 48 h assessing changes with treatment. a qRT-PCR of 45S (pre)-rRNA transcript at 24, 48 and 72 hrs (h), P < 0.015. b Silver staining of nucleolar organizer regions (NORs). c Immunostaining of Pol I, UBF, RRN3, Nucleolin, B23, and Fibrillarin (all green). d Cell cycle analysis of proliferating and TGFβ-treated NMuMG cells using the FUCCI technology, DAPI (blue), S/G2/M (geminin, green) and G1 (Cdt1, red), UBF (magenta) and merged. Colocalized green and red fluorescence indicates G1/S arrest (yellow). e rRNA synthesis (EU, red), Pol I (green) and merged (yellow) in the mouse neural crest. NT = neural tube, highlighted by white dotted box. f ChIP analysis of TIP5 binding to the rDNA, E-cadherin (Cdh1) and Snail1 promoters, P < 0.03. g HpaII methylation assay of the rDNA promoter, P < 0.007. h ChIP analysis of H3K4Me3 and H3K27Ac marks to the rDNA promoter, 18S rDNA and 28S rDNA, P < 0.006 and P < 0.005. i ChIP analysis of Pol I and UBF binding to rDNA promoter, 18S rDNA and 28S rDNA, P < 0.015 and P < 0.04. j ChIP analysis of SIRT7 binding to rDNA promoter, 18S rDNA and 28S rDNA, P < 0.005. k ChIP analysis of Snail1 binding to rDNA promoter, 18S rDNA, and 28S rDNA, P < 0.03. l Snail1 (green), E-cad (green), EU (red). Fibrillarin (green), UBF (green) and DAPI (blue) in proliferating (Control) and inducible Snail1 overexpression in NMuMG cells. Quantification of EU, P < 0.0002. All error bars ± SD, n = 3. Asterisk denotes t-test significance. Scale bar for all images = 50 µm