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. 2019 Apr 23;10:1897. doi: 10.1038/s41467-019-09810-w

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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. 10 — The R-point governs multiple programs of tumor suppression. a H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 μM 4-OHT for 0, 8, or 16 h. RNA was extracted from the cells, and gene expression patterns were analyzed by mRNA sequencing. Expression of genes 8 or 16 h after serum stimulation was quantified as log₂(fold change) relative to the average of control reactions (i.e., before serum stimulation, 0 h) for each cell line. Differential gene expression in response to expression of wild-type RUNX3 was analyzed by plotting log₂(E_{Rx3 + 16 h}/E_{Rx3 + 0 h}) and log₂(E_{vec + 16 h}/E_{vec + 0 h}); the results are shown on the left. Differential gene expression changes in response to expression of RUNX3-K94/171R were analyzed by plotting log₂(E_{Rx3KR + 16 h}/E_{Rx3KR + 0 h}) and log₂(E_{vec + 16 h}/E_{vec + 0 h}); the results are shown on the right. E_{Rx3 + 16 h} and E_{Rx3 + 0 h}, E_{Rx3KR + 16 h} and E_{Rx3KR + 0 h}, and E_{vec – 16 h} and E_{vec – 0 h} are the average expression levels of genes 0 or 16 h after 4-OHT stimulation in H460-ERT2-RUNX3, H460-ERT2-RUNX3-K94/171R, and H460-vec cells, respectively. Yellow spots indicate genes regulated in a RUNX3-independent manner. Red and blue spots indicate genes regulated in a RUNX3-dependent manner 8 and 16 h after 4-OHT stimulation, respectively (FDR < 0.001, p < 0.05). b RUNX3-dependent genes involved in various signaling pathways were analyzed using the DAVID Bioinformatics Resources 6.8³⁷. Gene expression levels 16 h after 4-OHT stimulation in the indicated cells were quantified as the fold change relative to the average of un-stimulated levels. Z-scores of log₂(E_{Rx3 + 16 h}/E_{Rx3 + 0 h}) and log₂(E_{Rx3KR + 16h}/E_{Rx3KR + 0 h}) for major signaling pathways are shown for H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells. Numbers at the top or bottom of the bars indicate the numbers of genes significantly up- or downregulated by inducer treatment. All categories were enriched with p < 0.05