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. 2015 Dec 25;291(8):4185–4196. doi: 10.1074/jbc.M115.705723

FIGURE 6.

FIGURE 6.

Enhanced expression of thermogenesis-related genes and lipolysis in the cultured primary brown adipocytes from Prip-KO mice. Cultured primary brown adipocytes from wild-type (WT) and Prip-KO (KO) mice were stimulated with 10 μm isoproterenol (Iso, +) or PBS (Vehicle, −) for 4 (B, F, and G) or 6 h (C–E and H). A, comparison of lipid metabolism-related proteins. Whole lysates from the cultured cells were analyzed by immunoblotting. Similar results were obtained from 3 independent experiments, and representative images are shown. B, expression of Ucp1 mRNA in the isoproterenol-stimulated cells was analyzed by qRT-PCR. The WT value in vehicle is set to 1. C and D, the expression of UCP1, PRIP1, and PRIP2, and the phosphorylation of HSL (p-HSL S660, p-HSL S563) and perilipin (p-perilipin S497) in cell lysates were analyzed by immunoblotting. Assessment of UCP1 protein expression is shown in C using β-actin as the loading control. Arrowheads in D indicate the specific PRIP1 bands. HSL and perilipin were used as the loading controls (D). Similar data were obtained from 3 independent experiments, and representative images are shown (C and D). Bands were quantified using ImageJ software. The mean value in WT is set to 1. E, brown adipocytes stimulated with or without isoproterenol were stained with Oil Red O. A set of typical images from 3 independent experiments is shown. Scale bar, 50 μm. Each lower panel shows a magnified view of the framed area in the upper panel. F–H, expression of Pgc1α, Pparα, Pparδ, Dio2, and Pparγ mRNA in the isoproterenol-stimulated cells was analyzed by qRT-PCR (F and H). The quantity in WT cells treated with vehicle is set to 1. Phosphorylation of CREB (p-CREB S133) and p38 MAPK (p-p38 MAPK T198) in cell lysates were analyzed by immunoblotting (G). Total amounts of CREB and p38 MAPK were used as the controls. Similar data were obtained from 3 independent experiments, and representative images are shown. The data represent the mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus the corresponding WT value.