Fig. 3: Ucp1-En4 and Ucp1-En6 regulate Ucp1 expression and affect mitochondrial function under cold stimulation. | Communications Biology

Fig. 3: Ucp1-En4 and Ucp1-En6 regulate Ucp1 expression and affect mitochondrial function under cold stimulation.

From: Identification of a distal enhancer of Ucp1 essential for thermogenesis and mitochondrial function in brown fat

Fig. 3

a qRT-PCR analysis of Ucp1 mRNA expression in brown adipocytes on day 8 of differentiation (n = 5). b Immunofluorescence staining of UCP1 (red) in brown adipocytes on day 8 of differentiation. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Quantification of UCP1-positive cells was performed by counting positive cells in five randomly chosen non-overlapping fields. Scale bar = 50 μm. c Western blot analysis of UCP1 in brown adipocytes on day 8 of differentiation (n = 4). Gray values of protein bands were quantified using Image J software, with Vinculin as a normalizer. d BODIPY (green) staining of lipid droplets in brown adipocytes on day 8 of differentiation. The nuclei were stained with DAPI (blue). The lipid sizes were measured as the diameter of individual lipid droplets in five randomly chosen non-overlapping fields. Scale bar = 50 μm. e qRT-PCR analysis of lipogenesis-related genes (Acly, Acaca, and Fasn) mRNA expression in brown adipocytes on day 8 of differentiation (n = 5). f Mitochondrial DNA (mtDNA) content in brown adipocytes on day 8 of differentiation (n = 5). g Western blot analysis of mitochondrial oxidative phosphorylation (OXPHOS) complex subunits in brown adipocytes on day 8 of differentiation (n = 4). Gray values of protein bands were quantified using Image J software, with Vinculin as a normalizer. On day 8 of differentiation, brown adipocytes were treated with 10 μM ISO or vehicle for 4 h before harvest for further experiments. Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t-tests.

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