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. 2018 Sep 13;8:13741. doi: 10.1038/s41598-018-32205-8

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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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Target sequence and structure of the PIPs targeting the GPR56 gene promoter and their effects on GPR56 expression. (a) Sequence comparison of the EVI1-binding sites between human and murine GPR56 promoter regions. The underlined sequence (−2378 to −2373 for human and −3059 to −3054 for murine) is a well-conserved binding sequence of the EVI1 transcription factor. (b) Chemical structure of the PI polyamide PIP/56-1 with its chemical formula and molecular weight. (c) Location of the target sequences of each PI polyamide in the human GPR56 promoter region is shown with each synthesized PIP. PIP/N5 covers −2389 to −2382 (tgatggat), PIP/56-1 covers −2381 to −2374 (acgGAAGA) of the EVI1-binding sequence, PIP/56-1 m is a mismatch PIP for PIP/56-1 with two nucleotide substitutions shown in red, PIP/56-2 covers −2377 to −2370 (AAGATaat) with the EVI1-binding sequence (underlined), and PIP/C-3 covers −2366 to −2359 (aattgggt). Red circles: Py N-methylpyrrole (Py); blue circles: Im N-methylimidazole (Im); β: β-alanine; Ac: Acetyl; Dp: N,N-dimethylaminopropylamine): γ-aminobutyric acid. (d,e) Expression of GPR56 in EVI1^high AML (UCSD/AML1 and Kasumi-3) measured by real-time RT-PCR 24 hours after the treatment with increasing concentrations (1 or 5 μM) of each PIP (AP2, 56-1, or 56-2). Parental cells were not treated (P) or were treated with the solvent of PIPs (0.1% DMSO) (C) as controls. Data represent the mean ± S.D. of triplicate determinations and are presented relative to control (parental). **P < 0.01 between parental control and treated cells (Student’s t-test). (h) The heat-map showing expression of 19 known EVI1 regulated genes in UCSD/AML cells treated with and without PIP/56-1. The expression of these 19 genes were reported to directly or indirectly regulated by EVI1^25–27. Heatmaps were generated in GeneSpring with microarray data from UCSD/AML cells with GPR/56-1 and control DMSO. Red and blue indicate up- and down-regulation for each gene, respectively. Asterisks (*) indicate significant differences between DMSO-treated and PIP/56-1-treated UCSD/AML cells (Student’s t-test, P < 0.05).