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. 2020 Sep 28;11:4882. doi: 10.1038/s41467-020-18648-6

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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. 3 — Naive CD4+ T cells were isolated from the spleen and differentiated into Th9 and Th17 cells for 5 days. ChIP assay and chromatin accessibility assay were performed on day 5. shRNA-expressing retrovirus was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a–c Kinetic analysis of chromatin accessibility, transcription factors, and chromatin modification markers binding on Il9 promoter locus in Th9 cells during Th9 differentiation. d FACS analysis of IL-9 and pSTAT5 expression in Th9 cells transduced with control (Scr), STAT5a-specific, or STAT5b-specific shRNA; cells were gated on transduced CD4+ live cells. e Chromatin accessibility analysis of Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus. f, g H3K27me3 modification and BATF binding at the Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus on day 5. h, i FACS analysis of IL-9 and IL-17 expression on day 5 in Th17 cells transduced with control vector or caSTAT5 retrovirus; cells were gated on transduced CD4+ live cells. j Chromatin accessibility analysis of Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. k The binding of BATF on Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. The p-values for a and b are compared to D0 for chromatin accessibility and STAT5 binding; D1 for other transcription factors and chromatin modification markers binding. One-way ANOVA with a post hoc Tukey’s test was used to generate p-values for all multiple comparisons in b. An unpaired two-tailed Student’s t-test was used for comparisons in a, d, e, h, j, and k. See also Supplementary Fig. 3.