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1
Iron Overload in Hereditary Tyrosinemia Type 1 Induces Liver
Injury through the Sp1/Tfr2/Hepcidin Axis
Wen-Dai Bao, Yao Fan, Yue-Zhen Deng, Ling-Yun Long, Jing-jing Wang,
Dong-Xian Guan, Zhen-Yu Qian, Peng An, Yuan-Yuan Feng, Zhi-Ying He,
Xiao-Fan Wang, H. Phillip Koeffler, Ronggui Hu, Jianshe Wang, Xin Wang,
Fudi Wang, Jing-Jing Li, Dong Xie
Table of contents
Supplementary materials and methods…………………………………..2
Supplementary Fig. 1………………………………………………………...6
Supplementary Fig. 2………………………………………………………...7
Supplementary Fig. 3………………………………………………………...9
Supplementary Fig. 4………………………………………………………..10
Supplementary Fig. 5………………………………………………………..11
Supplementary Fig. 6………………………………………………………..12
Supplementary Table 1……………………………………………….……..14
Supplementary Table 2……………………………………………….……..15
Supplementary Table 3……………………………………………….……..16
References……………..……………………………………………….……..18
2
Supplementary materials and methods
Hepatocyte isolation and treatment
Mouse primary hepatocytes were isolated by perusing livers using collagenase
type I (Worthington) in perfusion buffers. After digestion, the viable
hepatocytes were filtered through a cell strainer (BD Falcon) followed by two
centrifugations using cold DMEM medium supplemented with 10% FBS. The
purified hepatocytes were seeded in collagen-coated plates at 8×105 cells per
well in a 6-well plate.
For hepatocyte holo-transferrin (holo-Tf) treatment, the hepatocytes were
cultured in DMEM medium supplemented with 5% FBS and antibiotics for 3
hrs after isolation. Cells were treated with 0, 25 or 50µM holo-Tf
(Sigma-Aldrich) for 24 hrs in DMEM containing 2% FBS [4]. Hepatocytes were
washed with PBS, and harvested for RNA and protein extraction and analysis.
Reactive Oxygen Species (ROS) production of hepatocytes was detected
by ROS detection molecular probes CM-H2DCFDA according to the
manufacturer’s instruction (Invitrogen). Cells were treated with antioxidants
6hrs after isolation. After 24hrs treatment, hepatocytes were incubated with
these dyes at 37°C for 20 minutes before measurement of ROS production.
Cells were collected, and the staining signals were detected by a flow
cytometer using excitation sources and filters appropriate for fluorescein
(FITC).
3
Western Blot
Equal amounts of total cellular protein (15–30 mg) were resolved on
SDS-PAGE gels and transferred to polyvinylidene difluoride membranes
(Millipore, Bedford, MA). The membranes were blocked with 3% BSA for 1
hour and incubated overnight with primary antibodies. After washing, the
membranes were further incubated for 1 hour at room temperature with
corresponding horseradish peroxidase–conjugated secondary antibody then
washed 3 times. The immuno-reactive protein bands were visualized using an
enhanced chemiluminescence kit (Pierce Rockford, IL).
..
Preparation of Adenovirus
Full-length mouse open reading frame was amplified from a mouse liver cDNA
library by polymerase chain reaction (PCR) and inserted into the pcmv-myc
vector to obtain a myc tag first. The AdEasy Adenoviral Vector System
(Stratagene,Agilent Technologies) was used for the preparation of adenovirus,
the procedure was according to the manufacturer’s protocol. Sequences of
primers were available in Supplemental Materials. For animal experiment, we
intravenously delivered adenoviruses expressing Tfr2 or control virus, into 8
~10 week old female fah-/- mice after withdrawal of NTBC for one week.
Before the injection, we test the virus and minimize the amount of virus to
effectively overexpress the target gene but reduce the inflammation reaction.
4
For mouse primary hepatocytes transfection, the isolated cells were
transfected after a 6 h attachment. Adenoviruses were applied in 1ml fresh
medium at multiplicity of infection (M.O.I) of 1~4 for each cell in a 6-well dish
for 6hrs, then replaced with 2ml fresh medium. 48hrs after transfection,the
hepatocytes were harvested for RNA and protein extraction and analysis.
Luciferase reporter Assay
Cells were plated at a subconfluent density and co-transfected with 0.05µg of
the reporter plasmid, 0.5 µg of expression vectors, and 0.05 µg of Renilla
luciferase, pRL-TK as an internal control for transfection efficiency. Cell lysates
were prepared 48h after transfection, and the reporter activity was measured
using the Dual-luciferase reporter assay system (Promega). Transfections
were performed in triplicate and repeated three times to ensure reproducibility.
Chromatin Immunoprecipitation Assay
Sp1 binding sites were present in the Tfr2 promoter region (Figure 4D).The
sp1 antibody specific for ChIP (Santa Cruz Biotechnology) was used in this
assay. The detailed procedures were performed as previously described
[3].Sequences of primers are available in Supplemental Experimental
Procedures.
Small interference RNA for Sp1
Duplexes of the small interference RNA for Sp1 (RNAi-Sp1) were synthesized
5
and annealed (Shanghai GenePharma Co.,Ltd). The sequences of RNAi-Sp1
are 5-uugagucacccaaugagaatt-3 (forward) and
5-ttaacucaguggguuacucuu-3(reverse) [5], respectively, targeting both mouse
and human SP1 coding region relative to the translation start site. Huh7 and
hepa1-6 cells were transfected with Sp1 siRNA with lipofectamin RNAi max
reagent (invitrogen) according to the manufacturer’s protocol.
6
Supplementary Fig. 1. Iron accumulated in Fah-/- male mice after NTBC
withdrawal.
(A) Tissue iron concentrations of different organs (liver, heart, kidney intestine,
spleen) and (B) serum iron parameters were measured in male fah-/- mice
after withdrawal of NTBC for 4 weeks (n=6) (C) HE staining and Perls’
Prussian blue, Dab-enhanced perI’s iron staining of liver sections from the
male fah-/- murine liver withdrawal of NTBC for 4 weeks.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
7
Supplementary Fig. 2. Anti-oxidants could not rescue the
downregulation of hepcidin in fah-/- hepatocytes after NTBC withdrawal.
(A)ROS production in hepatocytes was examined by ROS-
sensitive fluorescence dye staining and calculated by FACS sorting.
Hepatocytes from wild-type mice were collected as control, hepatocytes from
fah-/- mice were treated with 1.0 mM NAC, 100μM PDTC or left untreated for
24 hours before staining.
(B) Hepatocytes from fah-/- mice withdrawal NTBC for one week were treated
8
with 1.0 mM NAC, 100μM PDTC for 0, 8, 16, 24 hours or left untreated
(control), all the cells were collected simultaneously after treatment. Hepcidin
mRNA level was determined by qRT-PCR and normalized to 18S expression.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
9
Supplementary Fig. 3. Add-back of NTBC restored Tfr2 expression in
fah-/- murine liver
(A) Protein level of Tfr2 in fah-/- murine liver of three groups: the control mice
with NTBC, the mice with one week of NTBC withdrawal and the mice with one
week add-back of NTBC after withdrawal (n=5). (B) Relative mRNA expression
level of Tfr2 in these mice.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
10
Supplementary Fig. 4. Tfr2 remained suppressed in fah-/- murine liver on
low iron diet following NTBC withdrawal
(A) Protein level of Tfr2 in fah-/- murine liver of three groups: the control mice
with NTBC, the mice with control food after four week of NTBC withdrawal and
the mice with low-iron food after four week of NTBC withdrawal (n=5). (B)
Relative mRNA expression level of Tfr2 in these mice.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
11
Supplementary Fig. 5. Iron status in fah-/- mice could not affect kidney
function.
(A and B) Crea(CR) and blood urea nitrogen (BUN) were determined as the
serum markers of kidney function in (A) fah-/- mice either with low iron or
control food intake after withdrawal of NTBC for 4 weeks and (B) in hfe-/-
×fah-/- , hjv-/-×fah-/- or fah-/- mice after withdrawal of NTBC for 5 weeks.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
12
Supplementary Fig. 6. Ros related genes alterations were restored by
low-iron diet, while the iron status in fah-/- mice could not affect tyrosine
catabolism genes expression.
(A~D) Relative mRNA expression level of (A) TAT, (B) HPD, (C) HGD, (D)
MaaI in fah-/- murine liver of three groups: the fah-/- mice with NTBC, the mice
with control food after four week of NTBC withdrawal and the mice with
low-iron food after four week of NTBC withdrawal (n=5). (E~H) Relative mRNA
expression level of (E) Nrf2, (F) Gsta1, (G) Gsta2, (H) Nqo1 in these mice. (I)
Protein levels of Nrf2 in these mice. (J) Total glutathione levels were measured
13
in these mice.
Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p
< 0.001
14
Supplementary Table 1. Serum iron of the HT1 patient
Serum iron(μg/dl)
Normal children
1 54.01±5.28
2 49.31±9.39
3 42.25±4.69
HT1 child 1 72.77±3.13
Serum iron were measured in the serum of the HT1 child patient and three
healthy children collected at the same day.
All samples were measured in triplicate for three times. Data are reported as
mean ± SEM,
15
Supplementary Table 2. List of Primers Used for Plasmids Construct
Plasmid Gene primers
Pcmv-myc Mouse Tfr2
CCGGAATTCggATGGAGCAACGTTGGGGTCTA
CGGGGTACCTCAAAAGTTATTGTCAATGTTC
pcDNA3.1 Mouse Sp1
CGGGGTACCATGAGCGACCAAGATCACTC
CCGCTCGAGGAAACCATTGCCACTGATATTA
pAd-track-CMV Mouse Tfr2
GGAAGATCTATGGCATCAATGCAGAAGCT
CCGCTCGAGTCAAAAGTTATTGTCAATGTTCC
Mouse Sp1
CGGGGTACCATGAGCGACCAAGATCACTC
CCCAAGCTTTCAATGGTGATGGTGATGATGA
16
Supplementary Table 3 List of Primers Used for qRT-PCR and CHIP
Gene Forward primer Reverse primer
18s-mus GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGCG
Hepcidin-mus AGAGCTGCAGCCTTTGCAC GAGGTCAGGATGTGGCTCTA
Tfr2-mus CCGCTATGGAGACGTGGTT TGGCGACACATACTGGGGACAG
Tfr1-mus CATGAGGGAAATCAATGATCG TA GCCCCAGAAGATATGTCGGAA
Fpn-mus TTGCAGGAGTCATTGCTGCTA TGGAGTTCTGCACACCATTGAT
Hfe-mus AGATCCGTGCCAAACAGAAC GGCGAGTCACTTTCACCAA
Hjv-mus TCTGACCTGAGTGAGACTGC GATGATGAGCGCTCCTACCTA
L-ferritin mus CCATCTGACCAACCTCCGC CGCTCAAAGAGATACTCGCC
HPD mus TCGGTGACCTTCTGGGTTG CAAAGCCCATCTTGTTGCAGTA
TAT mus TGCTGGATGTTCGCGTCAATA CGGCTTCACCTTCATGTTGTC
HGD mus ATTTGGGAATGAGTGTGCTTCA CTCAGCGTAGAGGTTGTAGGG
MaaI mus CCGGGAAGCCTATCCTCTACT CCGGGAAGCCTATCCTCTACT
Nrf2 mus TCTTGGAGTAAGTCGAGAAGTGT GTTGAAACTGAGCGAAAAAGGC
Gsta1 mus GACTGTGAGCTGAGTGGAGAAGAA CCGGCCATTGCAGCAA
Gsta2 mus CTCTTCAAACTCCACCCCTG TGGAGAAGAAGCCAGGACTC
Nqo1 mus AATGGGCCAGTACAATCAGG CCAGCCCTAAGGATCTCTCC
17
Sp1-mus GTGGTGATGGGATACATGATGAGA CAAGTGTGCTCGGAGATGTGA
Actin-hum GATCATTGCTCCTCCTGAGC ACTCCTGCTTGCTGATCCAC
Sp1-hum CCAGACCATTAACCTCAGTGCAT CTGGGCTGTTTTCTCCTTCCT
Tfr2-hum GGACGGTGGTGACTTTGGAA TGCTTCAGGACACTCTCAATGAG
tfr2-sp1-chip-1 GAAAAAAGAAGGGAAGAGGAAGG TCCAATCTCTTGTTCCCGACTC
tfr2-sp1-chip-2 GCTCAACCATCACACACAAACAC CTCGGCTTTCTACTTGCCTTCAT
tfr2-sp1-chip-3 CTTTTCTCAGGGCACATTCCAG GGTAAGAGAGAAGAGGGTAA
TGGGA
tfr2-sp1-chip-4 AATTTCTTAGGTTTTAGTGGGGTCA GGCCACACCCACTAGAGCAC
18
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