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. 2020 Jan 24;11:480. doi: 10.1038/s41467-019-13918-4

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© The Author(s) 2020

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 — a Workflow of murine primary cortical neurons (mPCN) isolation, drug treatment, and RT-qPCR analysis. b Epigenetic modulators used for treatment of mPCNs (left column) with enzymatic activities inhibited by them (right column). c Significant upregulation of paternal allele-derived Kcnk9 mRNA in DZnep-, SAHA-, VPA- and CI-994-treated E14 Kcnk9KO^mat mPCNs compared with DMSO-treated Kcnk9KO^mat mPCNs detected by RT-qPCR (3-day treatment, normalization to Impa2, DMSO n = 7 cultures/group, DZnep (20 µM) SAHA (30 µM), VPA (5 mM) and CI-994 (40 µM) n = 3 cultures/group). One-way ANOVA: F(5, 22) = 75.51, P < 0.0001; followed by Bonferroni’s multiple comparison post hoc test to Kcnk9KO^mat DMSO control, ****P < 0.0001. d Upregulation of paternal allele-derived Kcnk9 mRNA after treatment of Kcnk9KO^mat mPCNs with different CI-994 concentrations compared with paternal allele-derived Kcnk9 mRNA of DMSO-treated Kcnk9KO^mat mPCNs and Kcnk9 mRNA of DMSO-treated WT mPCNs detected by RT-qPCR (1 day treatment, normalization to Impa2, Kcnk9KO^mat DMSO n = 9; 4 µM n = 2; 20–80 µM n = 4 and WT DMSO n = 5 cultures/group). One-way ANOVA: F(5, 22) = 75.51, P < 0.0001; corrected with Bonferroni’s multiple comparison post hoc test to Kcnk9KO^mat DMSO mPCNs, ***P < 0.001****, P < 0.0001, c, d performed in 2–4 independent experiments. e Duration of Kcnk9 derepression in vitro. Workflow is illustrated in schematical representation (left). After 1 and 10 days, Kcnk9 is significantly upregulated after CI-994 treatment in mPCN compared with DMSO controls. One day DMSO n = 2, 1 day CI-994 n = 3, day 10 DMSO n = 6, day 10 CI-994; n = 4; n = samples (3–5 wells/sample; 6 embryos pooled). Day 1: P = 0.0297, day 10: P = 0.0001, Mann–Whitney U. c–e Values are means ± SEM. Statistical analyses and approaches are provided in Supplementary Table 1. Source data are provided as a Source Data file. Images displaying the mouse, the embryo and the neurons in this figure were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.