Classic browning agent CL316243 downregulates FoxO1 during adipose browning in mice.

(A–B) CL treatment downregulated FoxO1 and Smad3 in gonadal adipose tissue, evaluated by Western blotting (A) and densitometry (B), n = 6–8. (C) CL-treated mice show an increased mitochondrial content in gonadal adipose tissue, assessed by electron microscopy. (D–E) CL treatment upregulated mitochondrial proteins, evaluated by Western blotting (D) and densitometry (E), n = 6. (F–G) CL upregulated proteins importing iron into cells (DMT1 and TfR1) and mitochondria (Mfrn1), evaluated by Western blotting (F) and densitometry (G), n = 6. (H) Measurement of non-heme iron in gonadal adipose tissue (n = 6–8). (I) H&E staining of white adipose tissue (100x magnification). (J–K) Body composition analysis by NMR (n = 8–12). In all cases, mice treated with saline were used as the control (Ctrl) group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.