Supplementary Table 1. Primer Sequences Used for Quantitative Real-Time PCR Gene Forward Primer (5-3 ) Reverse Primer (5-3 ) cadl CTTGGGGGCGCGTCT CTGTTCTTTTGTGCCGTTTCG cyl-coenzyme Dehydrogenase, very long chain cadvl GGGGCGCCTTTGCGG GCGGCTCTGGGTTTG cyl-coenzyme Dehydrogenase, long chain CC GTCCCCGGGTGCCT GCCTGCTCCCGTGC COX-1 cetyl-coenzyme Carboxylase-1/α TCGGCCGCGTTCGG TCTCCGTCTGGGCGTGG CS cyl-coenzyme oxidase 1/ palmitoyl CCGCTCGGGTC CCGTGTGTTCCCCCGG cyl-co synthetase short-chain family member 2 NGPTL4 TCCCGCCCCCCTTC TGGTCTCTCCGTTGCC ap2 ngiopoietin-like 4 GGTGGGCTCTCCCT TCCGCCTTTCTCCTTCC TGL Fatty acid binding protein 4, adipocyte CTGGTCCCTTCCCCTC CCGCTGTGGGGGG CHOP-1 dipose triglyceride lipase GCGGCGGGTGGC CTGCGGTCGTCGGCC Cox5b C/EP homologous protein 1 TCGCCGGTGTGGG GTTGGGTTCCCGTTGGG CPT1a Cytochrome c Oxidase, Subunit Vb CTTGCGCTCTCGCTGGG GGCTTTCGCCCGGG CPT1b Carnitine palmitoyltransferase 1a, liver TGGGCTGGTCGTTGCTC TCGGGTTTGTCGGGG Carnitine palmitoyltransferase 1b, muscle Cyclin D1 TTGTGCTCTCCTGCCTC GGGTGGGTTGGTGCT DGT1 GCCCCTGCGTGTTTTGC CCTGGGTGTGCTCCC DGT2 Diacylglycerol O-acyltransferase 1 CCGGCCCTGCTCTT CTTCGGGTGCTGCGTTCTT FS Diacylglycerol O-acyltransferase 2 GGTCCCGGCGCTTCT GCCTGGTGGCTTCTGTGT FTP Fatty acid synthase CTGTGCCCCTGTTCCG CTCCCCGCCTTGGGG GDH Fatty acid transport protein TGCCCGTCCTGCCTC GCGGCCTTGGGGGTG Glyceraldehyde-3-phosphate dehydrogenase GLUT4 GGGGGGGCTTGCTG TGGGCCGTCCGTG GPT Glucose transporter 4 CGCCCGGCCGG TGCTGCTCGTCTTCTCGT Glycerol-3-phosphate acyltransferase, mitochondrial Gyk GTCGCCCGGGGCC CCCGGCTTTGGGGCT Hadha Glycerol kinase GCCCGTTCCGGG GGCCCCCCCTTTTGG Hydroxyacyl-Coenzyme dehydrogenase, alpha subunit
HSL TTCTCCGCCCTGCC TGTGGCTGGGCTTGTTG LCD Hormone-sensitive lipase CCCTGTTGCTCGGCTC CCGCCGTTCCCCTTGG LDLR Long-chain acyl-co dehydrogenase CTCCTGCTTCCGGTGCC CCCCTGTGCCTTGCTTG LPL Low density lipoprotein receptor GGCGGTCGGGTGTTG CGTTGTCTGGGGGTCTT PEPCK Lipoprotein lipase GCTTCCGCCGGTTC GGGCGGTCTGTCGTTCT Pfkl Phosphoenolpyruvate carboxykinase GCTCGCCCTTGCCT CTCCCGGTCCTCTGG PPRα Phosphofructokinase, liver TCGGCGCTTTCGGCTG GCCTTGTGCGGCGT Peroxisome proliferator activated receptor alpha PPRβ/δ TTGGCCCGTTCGGTTTG CGGTCTCCCCGTGTG Peroxisome proliferator activator receptor beta/delta PPRγ TGTGGGGTGCTCGGC CCGGCGTTGTCCCCTT Peroxisome proliferator activated receptor gamma Pref-1 CCCGGTGGCTTCGGTG GGGGGGGTCTCTTGTTGG Preadipocyte factor 1 Resistin GCCTTTCTTTCCCCTCCT GTCCGCTTTGCCTGTT SCD1 TTCTTGCGTCCTCTGGTGC CGGGTTGTGTTCTTGTCGT SREP-1c Stearoyl-Co desaturase-1 GGCCTCGCTCTCCG TCCTGCCTCTGTGCCTC Sterol regulatory element binding transcription factor 1c TKT TGGCTCCGGCTCTT TCCGCTTGTTTCCGC UCP2 Transketolase CGTGTGGTGGTCCGCT TTCCTCTCGTGCTGGTCTT UCP3 Uncoupling protein 2 GGTGGTGCCTCGCTC GCGTTCTGTTCGGGTCTTT Uncoupling protein 3 Visfatin CCCGTTGGTGGCTGT TGGTGCCGTGCCTCTG
Supplementary Figure 1. Effect of Smad3 deficiency on weight of various organs. C: Relative weight of liver (), quadriceps (), and brown adipose tissue (C) of 8-week-old and mice. Values represent percentage of body weight. D: Flow cytometry analysis of rdu-positive adipocytes in and epididymal adipose tissue. Values in bold represent percentage of rdu-positive adipocytes. and mice were injected intraperitoneally with 5 μg/g body weight of rdu for 3 consecutive days. dipocytes from epididymal adipose tissue were isolated, stained using rdu Flow kit and analyzed by flow cytometry as recommended by manufacturer (ecton Dickinson, Franklin Lakes, NJ). E: Representative hematoxylin-eosin stained paraffinembedded section of the and liver. Scale bars, 1 μm. F: Mean plasma cholesterol in and mice. Values represent mean ± SEM, n= 8/group. Liver weight (% body weight) D 4.6 4.4 4.2 4. 3.8 3.6 3.4 3.2 3. Muscle weight (% body weight).6.5.4.3.2.1. C rown fat weight (% body weight).35.3.25.2.15.1.5. rdu (FITC) 1 2 1 3 1 4 1 5 6.5 43. 3.3 1 2 1 3 1 4 1 5 6.2 47.7 3.7 E 5 1 15 2 7-D 25 5 1 15 2 F Cholesterol (mg/dl) 14 12 1 8 6 4 2 25 (x 1) (x 1) 7-D
Supplementary Figure 2. Values collected on the metabolic activity, food and water consumptions, and physical activity for 24-h dark-light cycle. F: The mean O 2 consumption (), CO 2 emission (), heat production (C), fuel consumption (D), and horizontal (E) and rearing (F) movements monitored over a 24-h light-dark cycle period. Horizontal movements (X amb ) and rearing movements (Z tot ) were measured by counting the number of horizontal and vertical infrared beam breaks. Respiratory exchange ratio (RER) was calculated by VO 2 /VCO 2. Values represent mean ± SEM, n= 8/group. 4 O consumption 2 4 CO emission 2 VO2 (ml/kg/h) 35 3 25 2 VCO2 (ml/kg/h) 35 3 25 2 15 1 C Heat (kcal/hr/kg) E Xamb (counts) 2 18 16 14 12 1 8 7 6 5 4 3 2 1 Metabolic rate 8 6 Horizontal movement D RER F Ztot (counts) 15 1 1.2 1.1 1..9.8 Fuel consumption.7.6 4 35 Vertical movement 3 25 2 15 1 5
Supplementary Figure 3. Effect of Smad3 deficiency on β-oxidation in liver. : Relative fold change in mrn level of the indicated genes in and liver as determined by real-time PCR. Values are normalized to ribosomal protein L27 (RPL27). Normalized values from mice were arbitrarily assigned a value of 1. Values represent mean ± SEM, n= 3/group. : The rate of [ 14 C]palmitate β-oxidation of hepatocytes freshly isolated from and liver. 2.5 cadl Hadha cox1 CS CPT1a PPRα PPRβ/δ [ 14 C]palmitate oxidation (nmol/1 x 1 5 cells) 2. 1.5 1..5.5 1 2 3 Relative fold-change in gene expression (gene/rpl27).
Supplementary Figure 4. Food intake and cholesterol level in and mice. and : Daily food intake () and mean food intake () by and mice on either high-fat diet () or low-fat diet () during and at the end of the 18- week feeding regime, respectively. Four experimental groups with a cohort of ten 8-week-old male mice each were assigned to either or (TestDiet, Richmond, IN) for 18 weeks. Group 1 and 3: and mice fed on (1% energy from fat, Yellow, 58Y2), respectively; Group 2 and 4: and mice fed on (45% energy from fat, Red, 58V8), respectively. C and D: Representative histological section (oil red O and methylene blue staining) of the skeletal muscle (C) and pancreas (D). Scale bars, 1 μm. (E) The mean plasma cholesterol in and mice on either or. Values represent mean ± SEM, n= 8/group. Daily food intake (g) Daily food intake (g) 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2. 2 4 6 8 1 12 14 16 18 Feeding period (wk) 2.5 2.45 2.4 2.35 2.3 2.25 2.2 2.15 2.1 2.5 2. C D E Cholesterol (mg/dl) 14 12 1 8 6 4 2