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. 2020 Jan 30;98(2):309–320. doi: 10.1007/s00109-019-01865-y

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — miR-181a-5p and its mRNA target Id2 conversely regulate Ifng expression in CD8⁺ T cells. RT-qPCR analysis of (a) signature genes Ifng and Tbx21 and (b) miR-181a-5p target genes Akt2, Id2, and Map2k1 upon transfection of cells with either miR-181a-5p or miR-451a mimics (n = 4). Values are relative to those of each experiment control condition where a nontargeting negative control miRNA Mimic was transfected. (c) Luciferase reporter assay to verify interaction between overexpressed microRNAs, miR-181a or miR-451a, and 3’ UTRs of selected targets: Id2, Map2k1 or Akt2. Mutation of miR-181a-5p binding sites in Id2 3’ UTR followed by luciferase assay was performed to prove miR-181a-binding specificity. Renilla/luciferase ratios were normalized to those obtained for empty pMig construct (n = 4). (d) RT-qPCR analysis of Id2, Ifng, Tbx21, Eomes, and Akt2 genes after siRNA-mediated Id2 knockdown (n = 4). Values are relative to those of each experiment control condition where a nontargeting negative control siRNA was transfected.* P ≤ 0.05