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. 2021 Dec 8;12:7144. doi: 10.1038/s41467-021-27442-x

Fig. 3. GPR180 is not a GPCR, but a component of TGFβ signalling pathway enhancing mature beige adipocytes function.

Fig. 3

a KEGG pathway analysis of differentially expressed genes (DEGs) of human beige adipocytes with ablated GPR180. Phosphorylation of SMAD3 at serine 423 in b non-starved human beige adipocytes after GPR180 silencing (n = 6; p = 0.0002), c beige adipocytes treated with different concentrations of TGFβ1 in combination with knockdown of GPR180 or TGFβR2 (ctrl siRNA vs siGPR180 p = 0.0416 for 1 pg/ml TGFβ1, p = 0.0129 for 100 pg/ml TGFβ1, p = 0.0034 for 1 ng/ml TGFβ1 and p = 0.0146 for 10 ng/ml TGFβ1) and d in white adipocytes overexpressing GPR180 (n = 6; p = 0.0050). Representative images of epitope tag immunostaining (green), nuclei stained by Hoechst (blue), e N-terminal HA tag and f C-terminal V5 tag in hMADS cells overexpressing modified GPR180; scale bar 100 µm. Experiment was performed 3 times with similar results. Long-term TGFβ1 treatment (72 h) dose-dependently promotes g UCP1 protein (n = 6; p = 0.0084 for 0.1 ng/ml and p < 0.0001 for 1 ng/ml) and h mitochondrial respiration (n = 5; cAMP uncoupled respiration p = 0.0020 for 1 pg/ml TGFβ1, p = 0.0334 for 10 pg/ml, p = 0.0008 for 100 pg/ml and p = 0.0012 for 1 ng/ml; maximal respiration p = 0.0205 for 1 pg/ml TGFβ1, p = 0.0178 for 10 pg/ml, p < 0.0001 for 100 pg/ml and p = 0.0127 for 1 ng/ml TGFβ1) in mature human beige adipocytes. Effect of i pharmacological (p = 0.0062 for 1 µM and p = 0.0017 for 10 µM) and j genetic (p = 0.0002) inhibition of TGFβR1 on UCP1 protein level in beige hMADS cells (n = 6). Effect of TGFβR2 silencing on k UCP1 expression (n = 6; p < 0.0001) and l HSL phosphorylation at serine 660 (n = 6; p = 0.0126). m Mitochondrial oxygen consumption rate following knockdown of individual TGFβ receptors in mature beige adipocytes (n = 5; p = 0.0082 for cAMP-stimulated uncoupled respiration and p < 0.0001 for maximal respiration). Effect of SMAD2 (p = 0.0001) and SMAD3 (p < 0.0001) knockdown on n UCP1 protein level (n = 6) and o mitochondrial respiration (n = 5; p = 0.0039 for SMAD3 and p = 0.0044 for SMAD2 cAMP-stimulated uncoupled respiration; p = 0.0001 for SMAD3 and p = 0.0011 for SMAD2 maximal respiration) in beige hMADS cells. Data are shown as average ±SEM. Statistical analysis was performed using two-sided Student´s t-test (b, j–l), one-way ANOVA with Dunnett’s post-hoc test (c, d, g, i, n) or two-way ANOVA with Tukey post-hoc test (h, m, o) and significance is indicated as *p < 0.05, **p < 0.01 and ***p < 0.001. cAMP cyclic adenosine monophosphate, GPR180 G protein-coupled receptor 180, HSL Hormone sensitive lipase, HSP90 Heat shock protein 90, OCR oxygen consumption rate, RFP red fluorescent protein, SMAD3 Mothers against decapentaplegic homolog 3, TGFβ1 Transforming growth factor β1, TGFβR1 Transforming growth factor β receptor type 1, TGFβR2 Transforming growth factor β receptor type 2, UCP1 Uncoupling protein 1.