miR-669a-5p promotes adipogenic differentiation and induces browning in preadipocytes

Figures

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  Figure 1.. Heatmap of C2MC expression in the mature 3T3-L1 adipocytes (day 10) and 3T3-L1 preadipocyte (day 2).

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  Figure 2.. miR-669a-5p is increased during differentiation of 3T3-L1 cells. (a)RT-qPCR to analyse the expression of miR-669a-5p in 3T3-L1 cells during differentiation, normalized to U6 expression. n = 3 per group.(b and c) RT-qPCR to analyse the mRNA expression levels of adipogenesis markers Pparγ and Fabp4 in 3T3-L1 cells during differentiation, normalized to β-actin expression. n = 3 per group.Data are representative of at least three individual experiments. Results are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus day 0 group.

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  Figure 3.. miR-669a-5p promotes the differentiation of 3T3-L1 cells. 3T3-L1 preadipocytes were transfected with mimic control or mimic miR-669a-5p (100 nM) on day 0 and day 4 after differentiation, cells were harvested on day 8 for analysis.(a) RT-qPCR to analyse the expression of miR-669a-5p in 3T3-L1 cells transfected with mimic control or mimic miR-669a-5p during differentiation, normalized to U6 expression. n = 3 per group.(b-c) Lipid accumulation was assessed by Oil Red O staining, and the absorbance was measured at 510 nm wave length. A representative image of three independent experiments is shown in B.(d) Lipid accumulation was assessed by intracellular TG content. n = 3 per group(e) RT-qPCR to analyse the mRNA levels of adipogenesis markers Pparγ, Fabp4 and perilipin2 in differentiated 3T3-L1 adipocytes transfected with mimic control or mimic miR-669a-5p, normalized to β-actin expression. n = 3 per group.(f) Western blot to evaluate the protein levers of adipogenesis markers PPARγ, FABP4 and PERILIPLIN2 in differentiated 3T3-L1 adipocytes transfected with mimic control or mimic miR-669a-5p, β-ACTIN was used as a loading control. n = 3 per group.(g) Quantitative densitometry of the…

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  Figure 4.. miR-669a-5p promotes adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 preadipocytes were transfected with mimic control or mimic miR-669a-5p (100 nM) on day 0 and day 3 after adipogenic differentiation, the cells were collected on day 6 for analysis.(a-c) RT-qPCR to analyse the expression of miR-669a-5p and brown fat-related genes (Pparγ and Pgc1α) during C3H10T1/2 differentiation, normalized to U6 or β-actin expression. n = 3 per group.(d-e) Lipid accumulation was assessed by Oil-Red O staining, and the absorbance was measuredat 510 nm wave length. A representative image of three independent experiments is shown in D.(f) Lipid accumulation was assessed by intracellular TG content. n = 3 per group(g) Western blot to evaluate the protein expression of brown fat genes PPARγ, PGC-1α and UCP1, β-ACTIN was used as a loading control. n = 3 per group.(h) Quantitative densitometry of the Western blots showed in F.(i) The quantification of mitochondrial DNA content. n = 3 per group(j) Mitochondrial mass was measured by MitoTracker Red staining on day 6. n = 3 per group.(k) Mitochondrial membrane potential was measured by TMRM on day 6. n = 3 per group.Data are representative of at…

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  Figure 5.. miR-669a-5p induces a brown adipocyte phenotype in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were transfected with mimic control or mimic miR-669a-5p (100 nm) on day 0 and day 4 after differentiation, cells were harvested on day 8 for analysis.(a) RT-qPCR to analyse the expression of thermogenic genes Pgc-1α and Ucp1 in 3T3-L1 adipocytes, normalized to β-actin expression. n = 3 per group.(b) Western blot to evaluate the protein levels of thermogenic genes PGC-1α and UCP1 in 3T3-L1 adipocytes, β-ACTIN was used as a loading control. n = 3 per group.(c) Quantitative densitometry of the Western blots showed in B.Data are representative of at least three individual experiments. Results are represented as mean ± SEM. *p < 0.05 versus mimic control group.

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  Figure 6.. miR-669a-5p enhances the mitochondrial biogenesis in 3T3-L1 cells. 3T3-L1 preadipocytes were transfected with mimic control or mimic miR-669a-5p (100 nM) on day 0 and day 4 after differentiation, the cells were collected on day 8 for analysis.(a) RT-qPCR analysis of mitochondrial genes involved in mitochondriogenesis, normalized to β-actin expression. n = 3 per group.(b) RT-qPCR analysis of mitochondrial genes involved in fatty acid uptake (Mito FA uptake) in mature adipocytes, normalized to β-actin expression. n = 3 per group.(c) RT-qPCR analysis of mitochondrial genes involved in β-oxidation (FAO) in mature adipocytes, normalized to β-actin expression. n = 3 per group.(d) RT-qPCR analysis of representative tricarboxylic acid (TCA) cycle genes in mature adipocytes, normalized to β-actin expression. n = 3 per group.(e) RT-qPCR analysis of representative genes of the five complexes of the mitochondrial electron transport chain (ETC) in mature adipocytes, normalized to β-actin expression. n = 3 per group.(f) The quantification of mitochondrial DNA content. n = 3 per group(g) Mitochondrial mass was measured by MitoTracker Red staining on day 8. n = 3 per group.(h) Mitochondrial…

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  Figure 7.. The expression of miR-669a-5p is upregulated in iWAT of mice exposed to cold. (a-b) RT-qPCR to analyse the expression of thermogenic genes Pgc-1α and Ucp1 in iWAT from mice at room temperature (RT) or post cold exposure, normalized to β-actin expression. n = 5 per group.(c) RT-qPCR to analyse the expression of miR-669a-5p in iWAT from mice at room temperature (RT) or post cold exposure, normalized to U6 expression. n = 5 per group.Data are representative of at least three individual experiments. Results are represented as mean ± SEM. *p < 0.05, **p < 0.01 versus RT group.