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. 2021 Feb 26;12:1333. doi: 10.1038/s41467-021-21594-6

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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 Metagene profiles of m⁶A site distribution along a normalized transcript containing three rescaled non-overlapping segments: 5′ UTR, CDS, and 3′ UTR in Ctrl and Mettl3^fl/flCd4-Cre SMARTA CD4⁺ T cells. b Pie chart showing the distribution of m⁶A sites in five regions of Ctrl and Mettl3^fl/flCd4-Cre SMARTA CD4⁺ T cells. c Bar chart depicting percentage of mRNAs with different internal m⁶A abundance. d Venn diagram showing the overlapping differentially expressed genes from RNA-seq and m⁶A-modified transcripts from m⁶A-miCLIP-SMARTer-seq. Heatmap of 208 differentially expressed genes with m⁶A modification is shown on the right. e Representative GO terms of the biological process categories enriched in differentially expressed transcripts with m⁶A peaks (Red: upregulated genes; blue: downregulated genes; gray: GO terms). f Integrative Genomics Viewer (IGV) tracks displaying RNA-seq (top panel) and m⁶A-miCLIP-SMARTer-seq (bottom panel) reads distribution of Tcf7 gene. The high-confidence m⁶A site is marked as a triangle. g RIP-qPCR analysis showing enrichment of METTL3 on Tcf7 mRNA in SMARTA CD4⁺ T cells. The Tcf7 mRNA enrichment is presented as IP/input and normalized to IgG group (n = 4 per group). h m⁶A-RIP-qPCR analysis of m⁶A enrichment on Tcf7 mRNA of Ctrl and Mettl3^fl/flCd4-Cre SMARTA CD4⁺ T cells (n = 4 per group). i Flow cytometry analysis of expression level of TCF-1 on T_FH cells on 8 dpi. Quantification of gMFI of TCF-1 is shown on the right (n = 5 per group). j Constructions of plasmids with wild-type or m⁶A site mutant in Tcf7 3′ UTR. k Luciferase reporter assay. Results were normalized to the luciferase activity of cells co-transfected with the pGL4.23 empty plasmid and EV-Myc plasmid (n = 6 per group). l RNA decay assay. The half-live of Tcf7 mRNA was detected by quantitative RT-PCR. The remaining mRNAs were normalized to t = 0 (n = 4 per group). m Quantitative RT-PCR analysis of Tcf7 mRNA abundance as in l, relative expression was normalized to t = 0 of Ctrl cells (n = 4 per group). Data are from one experiment with duplicate (a–e) or representative of at least three independent experiments (i–h, k–m). Error bars indicate standard error of the mean. P value was calculated by unpaired two-tailed Student’s t test (g–i) or two-way ANOVA coupled with multiple comparisons (k, m).