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. 2021 Jan 20;12:481. doi: 10.1038/s41467-020-20786-w

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© The Author(s) 2021

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. 6 — a The ranking of predicted transcriptional factors (TFs) capable of binding to RORCE was performed based on relative scores predicted with the JASPAR database (http://jaspar.genereg.net). b Red vertical line shows SOX-5 binding site (SOX-5-BS) in RORCE, which is very close to TS3. c, d A ChIP-qPCR assay was performed to determine the enrichment of SOX-5 in SOX-5-BS and RORγt promoter (RORγt p) in PI-stimulated EL4 cells (c) and WT Th17-polarized cells (d). e–h A ChIP-loop assay with an anti-SOX-5 antibody or control mouse IgG was performed to quantify relative interaction efficiency between the AS and TS3 mediated by SOX-5 in PI-stimulated EL4 (e), Th17-polarized (f), Th1-polarized (g), and Th2-polarized cells (h) from WT mice. i The sequence of SOX-5-BS (AACAA) in RORCE was deleted by using CRISPR/Cas9 technology (top). The knockout allele was confirmed by sequencing (bottom). j A ChIP-qPCR assay was performed to test the enrichment of SOX-5 in SOX-5-BS in Th17-polarized cells from SOX-5-BS-deficient (SOX-5-BS^−/−) or WT mice. k, l Quantitative analysis of 3C-qPCR (k) and ChIP-loop assays (l) evaluating the interaction between TS3 and AS in Th17-polarized cells from SOX-5-BS-deficient or WT mice was performed. Th17 D, Th1 D, and Th2 D represent Th17, Th1, or Th2 differentiation under the corresponding polarization conditions, respectively, and N represents naïve cells. Mean ± SEM are shown, n = 4 independent experiments, unpaired two-tailed Student’s t-test (b–h, j–l). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.