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. 2023 Feb 16;14:863. doi: 10.1038/s41467-023-36595-w

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© The Author(s) 2023

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. 2 — a IP-MS analysis showing the interacting partners of Nsun2 in induced Th17cells. b Co-immunoprecipitation showing the interaction of Nsun2 and RoRγt. c GST pull-down assay showing the interaction between Nsun2 and RoRγt. d PLA analysis for Nsun2 and RoRγt in induced Th17 cells. Scale bars, 5 μm. DAPI, nuclei. e The footprint profiles of RoRγt in Th17 cells with (Red) or without (Green) Nsun2 depletion. P value was determined by two-tailed Student’s t-test. f The metaplots of normalized RoRγt signal along gene bodies from Transcription Start Site (TSS) to Transcription Termination Site (TTS), plus or minus 3 kb. Genes had been divided into three groups including Nsun2 targets with (Red) or without (Gold) m⁵C and others (Gray). g, h Distribution of Nsun2 (g) or m⁵C (h) enrichment relative to RoRγt binding efficiency. The Nsun2 targets with m⁵C were divided into three equal groups according the normalized signal of RoRγt ChIP-seq at their TSSs (±3 kb), each group with 1035 genes. Using the associated IP and input samples, the overall Nsun2 or m⁵C enrichment distributed in each gene was respectively evaluated by MACS2. P value was determined by two-way Mann-Whitney U test and only value between Q1-1.5*IQR and Q3 + 1.5*IQR has been shown in boxplot. i Venn diagram showing the overlap between RoRγt targets and Nsun2 targets with m⁵C. j Heatmap displaying gene ontology biological processes enriched in the genes with m⁵C which were also bound by Nsun2 and RoRγt. P value was determined by right-sided Fisher test. k, l RT-qPCR showing the binding efficiency of Nsun2 and RoRγt on chromatin-associated Il17a (k) and Il17f (l) RNAs in induced Th17cells (n = 3 per group). m, n, m⁵C MeRIP-qPCR analysis showing the m⁵C enrichment in Il17a (m) and Il17f (n) mRNAs (n = 3 per group). IgG served as negative control. All data are representative of at least two independent experiments. Data are analyzed using two-tailed unpaired Student’s t-test. Error bars represent mean ± s.e.m. k–n Source data are provided as a Source Data file.