Online-Only Appendix (1)

Online-only appendix (1):

Figure legend

(A) Representative images of BAT and WAT sections stained with hemotoxylin and eosin (HE) from wild-type (+/+) and Cideb-null (-/-) mice at 22-weeks of age (n=5). Scale bars =50 µm.

(B&C) Relative mRNA levels of adipogenesis markers in BAT (B) and WAT (C) of wild-type and Cideb-null mice (n=3) under feeding condition by quantitative real-time PCR analysis, respectively.

(D). Fatty acid uptake in isolated primary hepatocytes from 3-month-old wild-type and Cideb-null male mice (n=5, each). For the measurement of fatty acid uptake in hepatocytes, 2 ×105 viable cells/well hepatocytes were cultured in 12-well plates for 24 h with DMEM containing 10% FCS. The reaction was carried out in triplicate (500 µl per well) each containing 22 µM [9,10(n)-3H]palmitate (1 μCi/well) HBSS with 0.5 mg/ml fatty acid-free BSA. After incubation at 37°C under humidified air containing

5% CO2, the monolayers were scraped to centrifuge tubes in 0.5 mL Dulbecco's PBS, and each well was washed twice with 0.5 mL Dulbecco's PBS. All DPBS were combined, and 5 mL hexane/isopropanol (3:/2) was added. After vigorously mixing, the tubes were centrifuged at 3,000×g for 5 min. The upper-phase containing lipid was removed to the vials, and dried under nitrogen steam. 10 mL scintillation solution was added, and the radioactivity was measured. The protein pellets were dried, and dissolved in 0.5 mL 0.2 M KOH solution overnight. The protein content was determined by Bradford method.

(E) Representative western blot analysis of cytochrome C (10μg /lane), Cox IV (10 μg /lane)and CPT2 (40 μg /lane) protein levels in liver, BAT, skeletal muscle (SM）and heart of wild-type and Cideb-null male mice (n=3 each) under fed state. (F) Plasma insulin concentrations during a glucose-tolerance test in wild-type and Cideb-null mice at 10 weeks of age (n=8 each). All data were represented as mean ±s.e.m value.

(G) Relative mRNA levels of glucokinase (GK), glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) in liver of wild-type and Cideb-null male mice (n=5) under feeding condition by quantitative real-time PCR analysis.