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. 2022 Aug 11;13:4711. doi: 10.1038/s41467-022-32322-z

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© The Author(s) 2022

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. 4 — a Heatmap of top ten downregulated TFs in circTmem241^−/− ILCPs versus circTmem241^+/+ ILCPs by transcriptome analysis. b Relative expression levels of top 10 TFs in circTmem241^+/+ and circTmem241^−/− ILCPs were analyzed by qRT-PCR. Fold changes were normalized to endogenous Gapdh. n = 3 biological independent experiments. c Top 10 downregulated TFs were knocked down in ILCPs were analyzed by FACS. n = 3 biological independent experiments. d Elk3 protein levels in circTmem241^+/+ or circTmem241^−/− ILCPs by western blotting. e Enrichment of circTmem241 on Elk3 gene promoter was analyzed by CHIRP assay. CircTmem241 probe was biotin labeled. n = 4 for each group. f Enrichment of circTmem241 on Elk3 gene promoter was further validated by dot blot. g Luciferase reporter assay was performed to validate circTmem241 function on Elk3 transcription activation. FL, full-length (represent −2000~0 region upstream Elk3 transcriptional start site). n = 4 for each group. h, i CircTmem241 hairpin region (HR) sequence mutations were incubated with Elk3 promoter regions, followed by dot blot. j DNase I assay of chromatin accessibility in Elk3 promoters of circTmem241^+/+ or circTmem241^−/− ILCPs by qRT-PCR. n = 3 for each group. k Transcription activities of Elk3 in circTmem241^+/+ or circTmem241^−/− ILCPs were measured by nuclear run-on assay. n = 4 for each group. l ILCPs from circTmem241^+/+ or circTmem241^−/− mice were infected with virus expressing circTmem241-WT or circTmem241-mut, followed by in vitro differentiation assay. ILC3s (CD3⁻CD19⁻CD127⁺CD45^loRORγt⁺) were analyzed by FACS. n = 5 for each group. WT, wild-type circTmem241; mut, circTmem241 mutation. *P < 0.05, **P < 0.01, and ***P < 0.001. Data were analyzed by an unpaired two-side Student’s t test and shown as means ± SD. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.