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. 2020 Apr 23;11(4):269. doi: 10.1038/s41419-020-2474-4

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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. 5 — a C-Jun overexpression upregulated miR-4476 confirmed by QPCR assays. b Schematic representation of the promoter regions of miR-4476 with the putative c-Jun TFBSs (site A and site B) and the structure of the wild-type (WT) and TFBS mutant (MutA, MutB, and MutAB) luciferase reporters driven by the promoter. c ChIP-QPCR and PCR gel showed there was significant c-Jun enrichment in the specific regions containing the two predicted binding sites. d EMSA result was shown from nuclear proteins extracted from U87 and LN229 cells after incubation with individual DIG-ddUTP-labelled oligonucleotide probes (lanes 2–6, 9–13). The free probe of labelled c-Jun was run in lanes 1 and 8 as a control. A 100-fold excess of unlabelled c-Jun-WT was used to compete with c-Jun binding (lanes 2 and 9, compared with lanes 6 and 13). A 100-fold excess of unlabeled mutated c-Jun-A and c-Jun-B was used to compete with binding of respective labelled probes (lanes 3–5 and lanes 10–12 compared with lanes 2 and 8). e Relative luciferase activity of the indicated promoter vectors in 293 T cells transfected with c-Jun plasmids. Mean ± S.D., *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.