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Supporting Materials
Supporting Materials and Methods
Construction of reporter gene plasmids
The mouse PNPLA3 gene promoter (-992 bp to +145 bp relative to the
transcription start site) was amplified by PCR and subcloned between the MluI and
XhoI sites of the luciferase reporter plasmid pGL3-basic vector (Promega); the
resulting reporter construct was designated as p-PNPLA3-992. To amplify different
promoter regions, the corresponding forward primers
5’-ACCACAAACCATCTAGCTCCCAG-3’ (p-PNPLA3-992),
5’-GCATCCAAGCCTAGGATTTGG-3’ (p-PNPLA3-170) and
5’-CGTCACCCGAAGACAGCTTAG-3’ (p-PNPLA3-43) were used with identifical
reverse primer 5’-CAGACATGCGTAAGCCCCGAC-3’. MluI and Xhol restriction
sites were added to each 5’ and 3’ primer, respectively. For site-specific mutations, the
putative SRE sites were mutated by PCR using the following primers:
5’-TGGTCCTAGAATGTTGGGGCCTC-3’ (p-PNPLA3-mut1) and
5’-GGGGCCTTTGCTTTTGTCACCCG-3’ (p-PNPLA3-mut2).
Transient transfection and luciferase assays
HepG2 cells were grown in 24-well plates using Dulbecco's Modified Eagle
Medium (GIBCO/BRL, 37°C, 5% CO2) containing 10% FBS. Cells were
co-transfected with the PNPLA3 promoter reporter plasmids and various expression
plasmids using LipofectamineTM 2000 (Invitrogen). Luciferase activity was measured
48 h later with the Dual Luciferase Reporter Assay System (Promega).
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Electrophoretic mobility shift assay (EMSA)
The LightShift Chemiluminescent EMSA Kit (Pierce) was used according to the
manufacturer’s instructions. HEK293 cells were cultured, and the nuclear proteins
were extracted according to the EMSA kit directions. The following biotin-labeled
oligonucleotides containing the PNPLA3 promoter SRE or the mutated SRE were
used:
5’-TATTTGCATGGTCCTAGTGGGGCGGGGCCTCAGCTGACGTCACCCGAAG
AC-3’ and
5'-TATTTGCATGGTCCTTGAATGTTAGGGCCTTTGCTAGCGTCACCCGAAGA
CA-3’, respectively.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed as previously described (26). The DNA fragments
were analyzed by PCR, and the promoter sequences spanning nucleotides -171 to +73
relative to the transcription start site were amplified using the following mouse
PNPLA3-specific primer pairs: 5’-AGCATCCAAGCCTAGGATTTGG-3’ (forward)
and 5’-CTCTGGGTCATACATGGTGCTTG-3’ (reverse).
Analytical procedures
Primary hepatocyte and hepatic concentrations of total triglycerides, total
cholesterol and free fatty acids were measured using enzymatic assay kits (Sigma) and
normalized to protein concentrations or sample weights. Serum ALT, AST, total
triglycerides, total cholesterol and free fatty acids were determined with enzymatic
assay kits (Sigma).
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For histological studies, livers were fixed in 4% paraformaldehyde and embedded
in paraffin. Then, 4-μm sections were cut and stained with hematoxylin and eosin. To
detect lipids, primary hepatocytes and 10-μm liver cryosections were fixed with
paraformaldehyde and stained with oil red O using 0.23% dye dissolved in 65%
isopropyl alcohol for 10 min.
Supporting Figures and tables
Supporting Figure S1. Adenovirus-mediated overexpression of PNPLA3 in the
mouse liver impaired insulin-induced Akt phosphorylation.
A, db/+ mice were treated as described in Fig. 7A. After 7 days, the lysates from
liver were subjected to western blot analysis using antibodies specific to Flag and
β-actin. B, C57BL/6J and db/+ mice were treated as described in Fig.7A.
Hematoxylin and eosin staining of liver sections (upper panel) and oil red O staining
of liver cryostat sections (down panel) from these mice. C, db/+ mice were treated as
described in Fig.7F. Liver protein lysates were subjected to western blot analysis.
Supporting Figure S2. Adenovirus-mediated knockdown of PNPLA3 in the
mouse liver minimally influenced the size and number of hepatic lipid droplets
ob/ob and db/db mice were treated as described in Fig.8A. Hematoxylin and eosin
staining of liver sections (upper panel) and oil red O staining of liver cryostat sections
(lower panel) from these mice.
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Supporting Table 1 Primers used in real-time PCR:
mouse SREBP-1c
5’-CTTCTGGAGACATCGCAAAC-3’ (forward)
5’-GGTAGACAACAGCCGCATC-3’ (reverse)
mouse PNPLA3
5’-ACGTGCTGGTGTCTGAGTTCC-3’(forward)
5’-AGGGACGTTGTCGCTCACTC-3’ (reverse)
mouse PNPLA2 (ATGL)
5’-TGTTTCAGACGGAGAGAACGTC-3’ (forward)
5’-TGAGAATGGGGACACTGTGATG-3’ (reverse)
mouse PNPLA5 (GS2-like)
5’-CGTCCCAAGCACCCTATTATG-3’ (forward)
5’-CCGCTCCACTTTCACCA-3’ (reverse)
mouse FAS (fatty acid synthase)
5’-CTTGGGTGCTGACTACAACC-3’ (forward)
5’-GCCCTCCCGTACACTCACTC-3’ (reverse)
mouse β-actin
5’–CCAGCCTTCCTTCTTGGGTAT-3’ (forward)
5’–TGCTGGAAGGTGGACAGTGAG-3’ (reverse)
mouse MTP
5’–GGGACTGGATGTGGCAGAG-3’ (forward)
5’–CACGCTGTCTTGCGGTTT-3’ (reverse)
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Supporting Table 2 Metabolic characteristics of 7-day–treated C57-BL/6J mice
with adenovirus expressing Ad-GFP (control) or Ad-PNPLA3
Ad-GFP
Ad-PNPLA3
Parameters
Body weight (g)
Liver/Body weight (100×)
23.90±0.83
26.41±1.95
6.13±0.26
6.01±0.75
Metabolic parameters
Liver triglyceride (mg/g of liver weight)
10.04±2.19
10.30±1.57
Liver cholesterol (mg/g of liver weight)
2.25±0.08
2.17±0.15
Liver free fatty acid (μm/g of liver weight)
9.44±0.68
9.69±0.69
Alanine aminotransferase (units/l)
536.92±301.6
500.37±199.6
Aspartate aminotransferase (units/l)
586.1±322.7
482.4±185.8
Triglyceride (mg/dl)
106.03±28.14
128.61±44.82
Blood parameters
Cholesterol (mg/dl)
Free fatty acid (mmol/l)
133.21±1.30
149.06±2.13
1.44±0.31
1.48±0.44
Data are means ±SE. C57-BL/6J mice (n= 5 per group).
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Supporting Table 3 Metabolic characteristics of 7-day–treated db/+ mice mice
with adenovirus expressing Ad-GFP (control) or Ad-PNPLA3.
Ad-GFP
Ad-PNPLA3
Parameters
Body weight (g)
Liver/Body weight (100×)
21.67±1.36
21.43±1.20
5.17±0.40
5.08±0.35
Metabolic parameters
Liver triglyceride (mg/g of liver weight)
7.36±1.30
7.53±1.15
Liver cholesterol (mg/g of liver weight)
2.31±0.14
2.38±0.11
Liver free fatty acid (μm/g of liver weight)
9.43±0.64
9.54±1.04
Alanine aminotransferase (units/l)
670.6±305.0
544.2±114.5
Aspartate aminotransferase (units/l)
656.9±256.6
481.5±112.0
Triglyceride (mg/dl)
94.74±13.92
138.35±16.63*
Blood parameters
Cholesterol (mg/dl)
Free fatty acid (mmol/l)
123.58±18.5
1.74±0.32
110.10±5.7
1.67±0.46
Data are means ±SE. db/+ mice (n= 5 per group), *P<0.05 versus Ad-GFP treated
db/+ control mice.
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Supporting Table 4 Metabolic characteristics of 7-day–treated ob/ob mice with
adenovirus Ad-siPNPLA3-scr (control) or Ad-siPNPLA3-2
Ad-siPNPLA3-scr
Ad-siPNPLA3-2
Parameters
Body weight (g)
50.43±4.00
51.97±2.20
9.20±0.61
8.61±1.39
Liver triglyceride (mg/g of liver weight)
149.08±21.33
128.72±24.64
Liver cholesterol (mg/g of liver weight)
18.49±0.71
18.20±0.84
Liver free fatty acid (μm/g of liver weight)
11.36±0.77
10.62±2.44
Alanine aminotransferase (units/l)
1311.6±601.9
1461.1±671.4
Aspartate aminotransferase (units/l)
1395.8±719.9
1478.0±758.8
Triglyceride (mg/dl)
49.14±3.82
53.13±2.76
Cholesterol (mg/dl)
241.88±3.99
237.62±3.42
0.80±0.42
0.81±0.12
Liver/Body weight (100×)
Metabolic parameters
Blood parameters
Free fatty acid (mmol/l)
Data are means ±SE. ob/ob mice(n= 4 per group).
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Supporting Table 5 Metabolic characteristics of 7-day–treated db/db mice with
adenovirus Ad-siPNPLA3-scr (control) or Ad-siPNPLA3-2
Ad-siPNPLA3-scr
Ad-siPNPLA3-2
Parameters
Body weight (g)
Liver/Body weight (100×)
46.86±3.96
45.92±7.78
7.97±0.48
8.84±1.16
Liver triglyceride (mg/g of liver weight)
73.45±10.72
67.92±14.15
Liver cholesterol (mg/g of liver weight)
18.37±2.72
17.67±1.81
Liver free fatty acid (μm/g of liver weight)
14.59±1.71
14.85±3.31
Alanine aminotransferase (units/l)
992.3±200.7
925.2±584.4
Aspartate aminotransferase (units/l)
1190.2±246.5
1064.1±617.4
Triglyceride (mg/dl)
133.99±16.67
120.72±8.87
Metabolic parameters
Blood parameters
Cholesterol (mg/dl)
Free fatty acid (mmol/l)
Data are means ±SE. db/db mice(n= 4 per group).
216.20±6.87
2.21±0.11
231.30±13.07
2.00±0.10