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. 2019 Aug 6;10:3536. doi: 10.1038/s41467-019-11302-w

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© The Author(s) 2019

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 — TonEBP suppresses Adrb3 gene expression. a Correlation of TonEBP mRNA with Adrb3 mRNA in human subcutaneous adipocytes (n = 15). b Adrb3 mRNA levels in iWAT from animals fed with a CD or HFD (CD, n = 5; HFD, n = 13). c Adrb3 immunostaining of iWAT from animals exposed to cold (4 °C). Scale bars, 100 μm. d Adrb3 mRNA abundance in primary adipocytes (n = 4). e mRNA abundance of thermogenic genes in 3T3-L1 adipocytes transfected with various siRNA’s as indicated (n = 4). f Schematic representation of the mouse Adrb3 gene promoter including the TonEBP binding site (TonE). A–C indicate regions for bisulfite sequencing (n = 4). g 3T3-L1 adipocytes were transfected with siRNA followed by an Adrb3 promoter-luciferase reporter construct. Luciferase was measured (n = 4). h TonEBP binding to the TonE (A region) of the Adrb3 promoter in 3T3-L1 adipocytes (n = 4). i The Adrb3 promoter-luciferase reporter described above (WT) and a mutant reporter where TonE was removed (ΔTonE) were analyzed in 3T3-L1 adipocytes (n = 4). Chromatin accessibility of A, B, C, and D regions (see f) (j), and ChIP assay for H3K4me1, H3K4me3, H3K27ac, H3K27me3 and normal rabbit IgG (k) for A and B regions, on the Adrb3 promoter region of 3T3-L1 adipocytes (n = 4). l DNA methylation analysis of the Adrb3 promoter using bisulfite sequencing in 3T3-L1 adipocytes. Left: bacterial clones without (open circles) or with methylation (solid circles) from representative experiments are shown. Right: % of DNA methylation, mean + s.d. (n = 4). m ChIP assay for DNMT1 on the Adrb3 promoter in 3T3-L1 adipocytes (n = 4). n Co-immunoprecipitation analyses of TonEBP and DNMT1 in 3T3-L1 adipocytes. n represents number of biologically independent samples (a, b, d) or independent experiments with triplicate (e, g–m). All data are presented as mean + s.e.m. (b, d) or + s.d. (e, g–m). AU arbitrary unit. The p-values are determined by unpaired t-test (d, j–l) or one-way ANOVA (b, e, g, h, m). ^#p < 0.05 vs. CD (a, b), scr siRNA (e), empty vector (g), anti-rabbit serum (h), WT (i), or anti-rabbit IgG (m). *p < 0.05 vs. +/+ (a–d), TonEBP siRNA (e), or scr siRNA (g–m). Source data are provided as a Source Data file