Fig 1: LncRNA FOXC2-AS1 may regulate white adipocyte browning through the autophagy signaling pathway. (A) Heatmap of differentially expressed genes between FOXC2-AS1 overexpression and NC groups in white adipocytes (n= 3 biological independent samples). (B) Pathway analysis of genes that were differentially expressed after FOXC2-AS1 overexpression in white adipocytes. (C, E) Protein levels of autophagy-related marker LC3 were detected by western blot after overexpression or knockdown of FOXC2-AS1 on day 4 and day 8 of differentiation. (D, F) Gray analysis of autophagy-related marker LC3 proteins and calculation of the ratio of LC3II to LC3I after overexpression or knockdown of FOXC2-AS1 on day 4 and day 8 of differentiation program. (G) Protein levels and gray analysis of UCP1 were evaluated in the differentiated adipocytes on day 4 from Si-FOXC2-AS1 groups treated with or without autophagy inhibitor 3-MA (5 mM) for additional 4 h. (H) OCRs of the differentiated adipocytes from Si-NC, Si-FOXC2-AS1 and Si-FOXC2-AS1 groups treated with 3-MA (5 mM) for additional 4h were evaluated. Quantitation of OCRs including basal respiration, ATP production, maximal respiration, proton leak, and spare respiration capacity. n=4 technical replicates per group. The representative immunoblotting graphs of western blot were shown of 3 biological independent samples. β-actin served as internal control. Values were presented as the mean ± SD. *P < 0.05 compared between the Si-NC and Si-FOXC2-AS1 groups. +P < 0.05 compared between Si-FOXC2-AS1 and Si-FOXC2-AS1 groups treated with 3-MA groups. “n.s.” means no significance.
Fig 2: Absence of BMP4 in PVAT produces a white-like phenotype. (A) Oil Red O staining of adipocytes differentiated from brown preadipocytes on day 8. (B) Western blot analysis of UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPβ in brown adipocytes with LacZ knockdown or BMP4 knockdown. (C) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in brown adipocytes with LacZ knockdown or BMP4 knockdown. (D)Schema of FABP4/aP2-Cre-BMP4flox/flox mouse models. (E) Gross picture of adult mouse thoracic aortic PVAT. (F) Representative HE and IHC(UCP1) microscopic picture of PVAT isolated from BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks. (G) Western blot analysis and quantification (H) of BMP4, UCP1 PGC1α, aP2, PPARγ and C/EBPβ in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 6–9). (I) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 7). (J) Schema of UCP1-Cre-BMP4flox/flox mouse models. (K) Representative HE and IHC(UCP1) microscopic picture of PVAT isolated from BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with WD for 16 weeks. (L) Western blot analysis and quantification (M) of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPβ in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with WD for 16 weeks (n = 6–9). (N) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with WD for 16 weeks (n = 7). Values are means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test (C, H, I, M and N). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: NONMMUT024512 and n281160 may involve in regulation of Pparα expression. (A) Representative microscopic images at day 0 and 4 of differentiation, and Oil red O staining at day 8. (B) UCP-1 immunofluorescence of primary brown adipocytes at day 8 of differentiation (original magnification, ×200). (C) Transcriptional levels of Ucp1, Pparα and two candidate lncRNAs at indicated times during the differentiation of brown adipocytes. (D–G) The expression of Pparα in adipocytes transfected with siRNAs targeting NONMMUT024512 or n281160 at day 6 of differentiation. Data are presented as the mean ± standard deviation (S.D.). Statistical significance evaluated by unpaired Student's t-test is reported as *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 4: BAT activity was increased in PZP TG mice. (A) Surface temperature of WT and PZP TG mice after refeeding time was measured by the infrared imager. (B,C) Immunoblots of total BAT lysates from WT and PZP TG mice showed UCP1 and PGC1a levels increased in PZP TG mice (n = 5). (D) Representative H&E staining images of BAT from WT and PZP TG mice show that PZP TG mice under IF conditions had smaller adipocytes and less lipid accumulation in the liver (scale bar = 100 μm). (E) Relative mRNA expression of adipogenic, thermogenic, and inflammatory genes in BAT from WT and PZP TG mice (n = 7).
Fig 5: FGF6 and FGF9 induce UCP1 expression by promoting prostaglandin E2 biosynthesis.a FGF6/9-induced Ucp1 expression in the presence or absence of heparin or HS. N = 3 per group. b Ucp1 expression in control (shLacZ) and Fgfr3 knockdown cell lines upon 24 h treatment with vehicle, FGF6, or FGF9. N = 3 per group. c Prostaglandin biosynthesis pathway. Genes encoding the enzymes marked in red are upregulated by FGF6 treatment. d Counts per millions for transcripts encoding PGE2 biosynthesis enzymes from RNA-sequencing of brown preadipocytes treated with FGF6 or vehicle for 24 h. e PGE2 concentration in the culture media of brown preadipocytes upon treatment with vehicle, FGF6, or FGF9. N = 3 per group. f Ucp1 expression in brown preadipocytes treated with PGE2 for 24 h. N = 2 per group. g Ucp1 expression in control (shLacZ) and Ptges knockdown cells treated with vehicle, FGF6, or FGF9 for 24 h. N = 3 per group. h Ucp1 expression in white preadipocytes treated with vehicle, FGF6, or FGF9 in the presence of PTGES-selective inhibitor (CAY10526) or equimolar concentration of DMSO. N = 3 per group. i Ucp1 expression in white preadipocytes treated with vehicle, FGF6, or FGF9 in the presence of both EP2 and EP4 receptor antagonists (AH6809 + AH23848) or equimolar concentration of DMSO. N = 3 per group. FGF6 and FGF9 were used at concentrations of 200 and 100 ng/ml, respectively. Data are presented as means ± SEM. Two-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. A representative from a total of two to three independent experiments is shown. Source data are provided as a Source Data file.
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