Supplementary Data

Home , ACADL, ACADVL, Fatty acid synthase, HADHA

SUPPLEMENTARY DATA

Supplementary Table 1. Primer Sequences Used for Quantitative Real-Time PCR

Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) Acadl CTTGGGAAGAGCAAGCGTACT CTGTTCTTTTGTGCCGTAATTCG Acyl-Coenzyme A Dehydrogenase, very long chain Acadvl GGAGGACGACACTTTGCAGG AGCGAGCATACTGGGTATTAGA Acyl-Coenzyme A Dehydrogenase, long chain ACC GTCCCCAGGGATGAACCAATA GCCATGCTCAACCAAAGTAGC Acetyl-Coenzyme A Carboxylase-1/α ACOX-1 TCGAAGCCAGCGTTACGAG ATCTCCGTCTGGGCGTAGG Acyl-Coenzyme A oxidase 1/ palmitoyl ACS AAACACGCTCAGGGAAAATCA ACCGTAGATGTATCCCCCAGG Acyl-CoA synthetase short-chain family member 2 ANGPTL4 TCCAACGCCACCCACTTAC TGAAGTCATCTCACAGTTGACCA Angiopoietin-like 4 aP2 AAGGTGAAGAGCATCATAACCCT TCACGCCTTTCATAACACATTCC Fatty acid binding protein 4, adipocyte ATGL CTGAGAATCACCATTCCCACATC CACAGCATGTAAGGGGGAGA Adipose triglyceride lipase CHOP-10 GAACGAGCGGAAAGTGGCA CATGCGGTCGATCAGAGCC C/EBP homologous protein 10 Cox5b ATCAGCAACAAGAGAATAGTGGG GTAATGGGTTCCACAGTTGGG Cytochrome c Oxidase, Subunit Vb CPT1a CTATGCGCTACTCGCTGAAGG GGCTTTCGACCCGAGAAGA Carnitine palmitoyltransferase 1a, liver CPT1b TGGGACTGGTCGATTGCATC TCAGGGTTTGTCGGAAGAAGAA Carnitine palmitoyltransferase 1b, muscle Cyclin D1 TTGTGCATCTACACTGACAACTC AGGGTGGGTTGGAAATGAACT DGAT1 GCCCCATGCGTGATTATTGC CACTGGAGTGATAGACTCAACCA Diacylglycerol O-acyltransferase 1 DGAT2 AACCGAGACACCATAGACTACTT CTTCAGGGTGACTGCGTTCTT Diacylglycerol O-acyltransferase 2 FAS AGAGATCCCGAGACGCTTCT GCCTGGTAGGCATTCTGTAGT Fatty acid synthase FATP CTGTAGCCAACCTGTTCCG CTCCCCGCCATAAATGAGGG Fatty acid transport protein GDH TGACCACAGTCCATGCCATC GACGGACACATTGGGGGTAG Glyceraldehyde-3-phosphate dehydrogenase GLUT4 GGAAGGAAAAGGGCTATGCTG TGAGGAACCGTCCAAGAATGA Glucose transporter 4 GPAT ACGCACACAAGGCACAGAG TGCTGCTCAGTACATTCTCAGTA Glycerol-3-phosphate acyltransferase, mitochondrial Gyk GTCAGCAACCAGAGGGAAACC CCACGGCATTATAGAGAGGCT Glycerol kinase Hadha AGCAACACGAATATCACAGGAAG AGGCACACCCACCATTTTGG Hydroxyacyl-Coenzyme A dehydrogenase, alpha subunit

HSL TTCTCCAAAGCACCTAGCCAA TGTGGAAAACTAAGGGCTTGTTG Hormone-sensitive lipase LCAD AACACCATGTATGCTCGGCTC CCGACCAAGATAATCCCCTTGAG Long-chain acyl-CoA dehydrogenase LDLR CTCCTGCATTCACGGTAGCC CCCACTGTGACACTTGAACTTG Low density lipoprotein receptor LPL GGACGGTAACGGGAATGTATG ACGTTGTCTAGGGGGTACTTAAA Lipoprotein lipase PEPCK AAGCATTCAACGCCAGGTTC GGGCGAGTCTGTCAGTTCAAT Phosphoenolpyruvate carboxykinase Pfkl GAACTACGCACACTTGACCAT CTCCAAAACAAAGGTCCTCTGG Phosphofructokinase, liver PPARα TCGGCGAACTATTCGGCTG GCACTTGTGAAAACGGCAGT Peroxisome proliferator activated receptor alpha PPARβ/δ TTGAGCCCAAGTTCGAGTTTG CGGTCTCCACACAGAATGATG Peroxisome proliferator activator receptor beta/delta PPARγ TGTGGGGATAAAGCATCAGGC CCGGCAGTTAAGATCACACCTAT Peroxisome proliferator activated receptor gamma Pref-1 CCCAGGTGAGCTTCGAGTG GGAGAGGGGTACTCTTGTTGAG Preadipocyte factor 1 Resistin AAGAACCTTTCATTTCCCCTCCT GTCCAGCAATTTAAGCCAATGTT SCD1 TTCTTGCGATACACTCTGGTGC CGGGATTGAATGTTCTTGTCGT Stearoyl-CoA desaturase-1 SREBP-1c AGGCCATCGACTACATCCG TCCATAGACACATCTGTGCCTC Sterol regulatory element binding transcription factor 1c TKT TGGCATACACAGGCAAATACTT TCCAGCTTGTAAATTCCAGCAA Transketolase UCP2 CAGATGTGGTAAAGGTCCGCT TTCCTCTCGTGCAATGGTCTT Uncoupling protein 2 UCP3 GAGATGGTGACCTACGACATCA GCGTTCATGTATCGGGTCTTTA Uncoupling protein 3 Visfatin CCCGATTGAAGTAAAGGCTGT TGGTAAGCCAGTAGCACTCTG

Supplementary Figure 1. Effect of Smad3 deficiency on weight of various organs. A–C: Relative weight of liver (A), quadriceps (B), and brown adipose tissue (C) of 8-week-old WT and KO mice. Values represent percentage of body weight. D: Flow cytometry analysis of BrdU-positive adipocytes in WT and KO epididymal adipose tissue. Values in bold represent percentage of BrdU-positive adipocytes. WT and KO mice were injected intraperitoneally with 500 μg/g body weight of BrdU for 3 consecutive days. Adipocytes from epididymal adipose tissue were isolated, stained using BrdU Flow kit and analyzed by flow cytometry as recommended by manufacturer (Becton Dickinson, Franklin Lakes, NJ). E: Representative hematoxylin-eosin stained paraffin- embedded section of the WT and KO liver. Scale bars, 100 μm. F: Mean plasma cholesterol in WT and KO mice. Values represent mean ± SEM, n= 8/group.

A B C 4.6 0.6 0.35 4.4 0.5 0.30 4.2 0.25 4.0 0.4 0.20 3.8 0.3 0.15 3.6 0.2

Liver weight 0.10

3.4 Muscle weight (% body weight) (% body weight) (% body weight) 3.2 0.1 fat weight Brown 0.05 3.0 0.0 0.00 WT KO WT KO WT KO D WT 6.5 KO 6.2 5 5

43.0 3.3 10 47.7 3.7 10 4 4 10 10 3 3 BrdU (FITC) 10 10 2 2 10 10

50 100 150 200 250 50100 150 200 250 (x 1000) 7-AAD (x 1000) 7-AAD E F 140 120 100 80 60 40

WT KO Cholesterol (mg/dl) 20 0 WT KO

Supplementary Figure 2. Values collected on the metabolic activity, food and water consumptions, and physical activity for 24-h dark-light cycle. A–F: The mean O2 consumption (A), CO2 emission (B), heat production (C), fuel consumption (D), and horizontal (E) and rearing (F) movements monitored over a 24-h light-dark cycle period. Horizontal movements (Xamb) and rearing movements (Ztot) were measured by counting the number of horizontal and vertical infrared beam breaks. Respiratory exchange ratio (RER) was calculated by VO2/VCO2. Values represent mean ± SEM, n= 8/group. A B

O2 consumption CO2 emission Day Night Day Night 4000 4000

3500 3500

3000 3000

2500 2500 (ml/kg/h) (ml/kg/h) 2 2 2000 2000 VO VCO

1500 WT 1500 WT KO KO 1000 1000 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 C Time (h) D Time (h) Metabolic rate Fuel consumption Day Night Day Night 20 1.2

18 1.1

16 1.0 14 0.9

12 RER 0.8

Heat (kcal/hr/kg) 10 0.7 8 WT WT KO KO 6 0.6 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 E Time (h) F Time (h) Horizontal movement Vertical movement Day Night Day Night 800 WT 400 WT KO 700 350 KO 600 300 500 250 400 200 300 150 Ztot (counts) Xamb (counts) 200 100 100 50 0 0 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 0.0 2.3 4.5 6.8 9.0 11.3 13.5 15.8 18.0 20.3 22.5 Time (h) Time (h)

Supplementary Figure 3. Effect of Smad3 deficiency on β-oxidation in liver. A: Relative fold change in mRNA level of the indicated genes in WT and KO liver as determined by real-time PCR. Values are normalized to ribosomal protein L27 (RPL27). Normalized values from WT mice were arbitrarily assigned a value of 1. Values represent mean ± SEM, n= 3/group. B: The rate of [14C]palmitate β-oxidation of hepatocytes freshly isolated from WT and KO liver. A B 2.5

Acadl WT 2.0 Hadha KO cells) Acox1 5 1.5 ACS CPT1a 1.0 PPARα C]palmitate oxidation β/δ (nmol/1 x 10 0.5

PPAR 14 [ 0 0.5 12 3 0.0 Relative fold-change WT KO in gene expression (gene/RPL27)

Supplementary Figure 4. Food intake and cholesterol level in A WT HFD 2.7 WT LFD WT and KO mice. A and B: Daily food intake (A) and mean KO HFD food intake (B) by WT and KO mice on either high-fat diet 2.6 KO LFD (HFD) or low-fat diet (LFD) during and at the end of the 18- 2.5 week feeding regime, respectively. Four experimental groups 2.4 with a cohort of ten 8-week-old male mice each were 2.3 assigned to either HFD or LFD (TestDiet®, Richmond, IN) 2.2 for 18 weeks. Group 1 and 3: WT and KO mice fed on LFD (10% energy from fat, Yellow, 58Y2), respectively; Group 2 Daily food intake (g) 2.1 2.0 and 4: WT and KO mice fed on HFD (45% energy from fat, 0 2 4 6 8 10 12 14 16 18 Red, 58V8), respectively. C and D: Representative Feeding period (wk) histological section (oil red O and methylene blue staining) of B the skeletal muscle (C) and pancreas (D). Scale bars, 100 μm. 2.50 WT 2.45 KO (E) The mean plasma cholesterol in WT and KO mice on 2.40 either HFD or LFD. Values represent mean ± SEM, n= 2.35 8/group. 2.30 2.25 2.20 2.15 2.10 Daily food intake (g) 2.05 2.00 LFD HFD

C WT KO

LFD

HFD

D WT KO

LFD

HFD

E 140 WT KO 120 100 80 60 40

Cholesterol (mg/dl) 20 0 LFD HFD