SUPPLEMENTAL TABLES
Supplemental Table 1. Characteristics of the diets.
Chow diet / MCD diet / Western dietReference / Picolab Rodent diet 20, #5053 / MP Biomedicals, #960439 / TD.120330, 22% HVO + 0.2% cholesterol diet, Teklad Research
Kcal/g of diet / 4.7 / 4.5 / 4.6
% Fat as calories / 13 / 20 / 45.3
% CH as calories / 62 / 58 / 37
% Proteins as calories / 24 / 22 / 17.7
Supplemental Table 2. Composition of the experimental diets.
Reference / MP Biomedicals, #960439 / TD.120330, 22% HVO + 0.2% cholesterol diet, Teklad Research
Formula (g/Kg) / Sucrose (455.3)
Corn Starch (200.0)
Corn Oil (100.0)
Alphacel Non-Nutritive Bulk (30.0)
Mineral Mix, AIN-76, (35.0)
Calcium Phosphate, dibasic (3.0)
L-Alanine (3.5)
L-Arginine HCl (12.1)
L-Asparagine Monohydrate (6.0)
L-Aspartic Acid (3.5)
L-Cystine (3.5)
L-Glutamic Acid (40.0)
Glycine (23.3)
L-Histidine HCl (4.5)
L-Isoleucine (8.2)
L-Leucine (11.1)
L-Lysine HCl (18.0)
L-Phenylalanine (7.5)
L-Proline (3.5)
L-Serine (3.5)
L-Threonine (8.2)
L-Tryptophan (1.8)
L-Tyrosine (5.0)
L-Valine (8.2)
Vitamin E, DL-alpha tocopherol acetate (250 IU/g) (0.484)
Vitamin A Palmitate (250,000 IU/g) (0.0792)
Vitamin D3, cholecalciferol (400,000 IU/g) (0.0055)
Ethoxyquin (0.02)
Biotin (0.0004)
Calcium Patothenate (0.0661)
Folic acid (0.002)
Inositol (0.1101)
Menadione (0.0496)
Niacin (0.0991)
p-Aminobenzoic acid (0.1101)
Pyridoxine HCl (0.0220)
Riboflacin (0.022)
Thiamine HCl (0.022)
Vitamin B12 (0.1% trit.) (0.0297)
Ascorbic acid (1.0166)
Corn Starch (3.4503) / Casein (230.0)
DL-Methionine (3.4)
Sucrose, fine ground (211.7098)
Corn Starch (80.0)
Maltodextrin (140.0)
Vegetable Shortening, hydrogenated (Primex) (220.0)
Soybean Oil (10.0)
Cholesterol (2.0)
Cellulose (50.0)
Mineral Mix, AIN-93G-MX (94046) (46.0)
Calcium Phosphate, dibasic (3.3)
Niacin (0.042)
Calcium Pantothenate (0.0224)
Pyridoxine HCl (0.0098)
Thiamin HCl (0.0084)
Riboflavin (0.0084)
Folic Acid (0.0028)
Biotin (0.0003)
Vitamin B12 (0.1% in mannitol) (0.035)
Vitamin E, DL-alpha tocopherol acetate (500 IU/g) (0.1)
Vitamin A Palmitate (500,000 IU/g) (0.0112)
Vitamin D3, cholecalciferol (500,000 IU/g) (0.0028)
Vitamin K1, phylloquinone (0.0011)
Choline Bitartrate (3.3)
TBHQ, antioxidant (0.046)
*This diet is a modification of TD.06303 to add 0.2% cholesterol. We also add 42 g/L glucose and fructose in the drinking water (55% fructose and 45% glucose, w/w).
Supplemental Table 3 – Primaries Antibodies for Immunohistochemistry.
a-SMA / Rabbit / Abcam / Ab32575 / Citrate / 1:400
Collagen 1a / Rabbit / Abcam / Ab292 / Citrate / 1:300
Desmin / Rabbit / Abcam / Ab15200 / Citrate / 1:400
K19 / Rat / Develpmental Studies Hybridoma Bank / Troma III / Pepsin + EDTA / 1:150
4-Hydroxynonenal / Rabbit / Abcam / Ab46545 / Citrate / 1:250
a-SMA, alpha smooth muscle actin; K19, Keratin 19.
Supplemental Table 4 – RT-PCR primers for analysis.
S9 / GACTCCGGAACAAACGTGAGGT / CTTCATCTTGCCCTCGTCCA
PANK1 v.1 / GAGAGAGTCCATCAGCAAAG / GAGAAAGTTCCCAACAAACAC
PANK1 v.2 / CTTATCTGAGCCTAACTCCATTC / CCACCGATATCCATACCAAAC
PANK2 / CTCTGCTTCTGGTGAACATC / GACAGCAGAGACCAAAGAAA
PANK3 / AGGAGGTGGTGCTTACAA / CCATTGAAACTGACGGAATCTA
PANK4 / GTGGAGCCTACAAGTTCAAG / CTCATGCGGGATGTTCTTC
PPCS / CCTGAACACAAGATCCACTC / CTCCAGCTTAAAGGAAACTACA
PPCDC / AACAACAGAGAGAGCCAAAC / GCAGGTCAATGTGGAGAAC
COASY / CCATCAATAGGAAGGTCCTG / CACAGCCACATCCATCTC
Vanin-1 / ATCCCAGACCCTCAAGTAAA / CATGTTCGCCACAACATAGA
Vanin-3 / GAGGCTTTGCTCCTGATAAA / CTGGTGAAGATCCAACCATAG
a-SMA / GTGGGGGACGAAGCGCAGAG / GGCCTTAGGGTTCAGCGGCG
Col1a1 / TTCCCTGGACCTAAGGGTACT / TTGAGCTCCAGCTTCGCC
Desmin / TACACCTGCGAGATTGATGC / ACATCCAAGGCCATCTTCAC
K-7 / TAGAGTCCAGCATCGCAGAG / CACAGGTCCCATTCCGTC
K-19 / GTGAAGATCCGCGACTGGT / AGGCGAGCATTGTCAATCTG
Caspase-2 / CAATGCTAACTGTCCAAGTCTA / GGGATTGTGTGTGGTTCTT
SOD-1 / GAGACCTGGGCAATGTGACT / GTTTACTGCGCAATCCCAAT
SOD-2 / CCGAGGAGAAGTACCACGAG / GCTTGATAGCCTCCAGCAAC
GSS / GCCTCCTACATCCTCATGGA / CCACATGCTTGTTCATCACC
GPx-1 / ACTACACCGAGATGAACGA / GACGTACTTGAGGGAATTCAG
Catalase / AGATGGAGAGGCAGTCTATT / AGATCTCGGAGGCCATAAT
Heme Oxigenase / GCTCGAATGAACACTCTGG / GTTCCTCTGTCAGCATCAC
PANK, Pantothenate kinase; PPCS, 4-Phosphopantothenoylcysteine synthase; PPCDC, Phosphopantothenoylcysteine carboxylase; COASY, CoA synthase; a-SMA, a-Smooth muscle actin; Col1a1, Collagen-a1; K7, Keratin 7; K19, Keratin 19; SOD-1 and -2, Superoxide Dismutase-1 and-2; GSS, Glutathione Synthetase; GPx1, Glutathione Peroxidase.
SUPPLEMENTAL FIGURES
Supplemental Figure 1 – Mice with Western diet-induced NASH show perturbations of CoA metabolism
A. Free CoA liver content in WT mice fed chow (n=8) or Western diet (n=8). B. Liver PANK-1 expression analyzed by Western blot. C. qRT-PCR analysis of whole liver mRNA for genes in the CoA synthetic pathway and CoA degradation pathway, in the same mice as A. Results were graphed as mean±SEM and normalized to expression in chow-diet fed mice. * P<0.05, ** P<0.01 chow versus Western diet.
Supplemental Figure 2 – Effects of Pantothenate and N-acetylcysteine in hepatic steatosis and inflammation
A. Representative photos from liver H&E staining in mice fed chow diet or MCD diet treated either with placebo, pantothenate and N-acetylcysteine in conjunction or in isolation. B. Quantification of liver triglycerides content. C. qRT-PCR analysis in whole liver for inflammatory markers. Results were normalized for chow-diet mice and graphed as mean±SEM. ** P<0.01 versus chow diet. # P<0.05 and ## P<0.01 versus MCD diet.
Supplemental Figure 3 – Treatment with Pantothenate and N-acetylcysteine does not decrease MCD-induced oxidative stress
A. qRT-PCR analysis in whole liver for enzymes involved in oxidative stress in mice fed MCD diet and treated with pantothenate and N-acetylcysteine. B. Liver sections from representative mice stained for the oxidative stress marker 4-hydroxynonenal (4-HNE) and respective morphometry. C. TBARS assay for all groups. Results were normalized for chow-diet mice and graphed as mean±SEM. * P<0.05, ** P<0.01 versus chow diet.