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. 2018 May 25;9:2080. doi: 10.1038/s41467-018-04455-7

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© The Author(s) 2018

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. 4 — The DIPG H3.3K27M mutation reduces the Cbx7 level bound to chromatin, but has no effect on Ezh2. a Co-immunostaining of FLAG (yellow) and H3K27me3 (green) in HEK293T cells expressing HaloTag-Ezh2/H3.3-FLAG, HaloTag-Ezh2/H3.3K27M-FLAG, HaloTag-Cbx7/H3.3-FLAG, or HaloTag-Cbx7/H3.3K27M-FLAG. Quantification analysis showed that the H3K27me3 level in HEK293T cells expressing H3.3K27M-FLAG is ∼7.0-fold lower compared to that expressing H3.3-FLAG (Supplementary Fig. 2). Scale bar, 5.0 µm. b Cumulative distribution of displacements for HaloTag-Ezh2 in HEK293T cells expressing H3.3-FLAG (N = 54 cells, n = 9034 displacements) or H3.3K27M-FLAG (N = 127 cells, n = 22,273 displacements), and for HaloTag-Cbx7 in HEK293T cells expressing H3.3-FLAG (N = 78 cells, n = 14,101 displacements) or H3.3K27M-FLAG (N = 89 cells, n = 15,755 displacements). The distributions were decomposed into three populations. Fitted parameters are shown in Supplementary Table 5. c Fraction of the chromatin-bound population (F₁) for HaloTag-Ezh2 or HaloTag-Cbx7 in HEK293T cells expressing H3.3-FLAG or H3.3K27M-FLAG. The data were obtained from Fig. 4b. Results are means ± SD. d Immunostaining of H3K27me3 in patient-derived SF8628 and control brain tumor 9427 cells by using antibody directed against H3K27me3 (green). DNA was stained with hoechst (red). Overlay images are shown. The H3K27me3 level in 9427 cells was ∼2.0-fold higher than that in SF8628 cells (Supplementary Fig. 2). Scale bar, 5.0 µm. e Cumulative distribution of displacements for HaloTag-Ezh2 in 9427 (N = 73 cells, n = 6806 displacements) and SF8628 (N = 43 cells, n = 6900 displacements) cells, and for HaloTag-Cbx7 in 9427 (N = 37 cells, n = 5280 displacements) and SF8628 (N = 32 cells, n = 8181 displacements) cells. The histograms were fitted with three components. Fitted data are shown in Supplementary Table 5. f Fraction of the chromatin-bound population (F₁) for HaloTag-Ezh2 in 9427 and SF8628 cells and for HaloTag-Cbx7 in 9427 and SF8628 cells. The data were obtained from e. Results are means ± SD from three biological replicates