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Supplemental Figures:
A
B
25
5
20
*
15
**
**
**
10
5
MDA level
(nmol/mg protein)
ROS
(pmol DCF formed/min/mg protein)
Fig. 1
0
4
3
*
**
2
**
1
0
Control
D-gal
5 mg/kg 1 mg/kg D-gal+NAC
Control
D-gal
5 mg/kg 1 mg/kg D-gal+NAC
D-gal+FGF21
D-gal+FGF21
C
D
80
80
60
*
*
*
40
**
20
CAT activity
(U/mg protein)
SOD activity
（ U/mg protein）
100
60
Control
**
40
20
D-gal
Control
5 mg/kg 1 mg/kg D-gal+NAC
D-gal
5 mg/kg 1 mg/kg D-gal+NAC
D-gal+FGF21
D-gal+FGF21
E
F
150
1.5
**
*
*
50
T-AOC
(U/mg protein)
***
100
**
**
**
0
0
GSH-Px activity
(U/mg protein)
**
0
Control
D-gal
5 mg/kg 1 mg/kg D-gal+NAC
***
1.0
***
*
0.5
0.0
Control
D-gal
5 mg/kg 1 mg/kg D-gal+NAC
D-gal+FGF21
D-gal+FGF21
Supplemental Fig. 1. Effect of FGF21 on the production of ROS and the parameters
of oxidative stress in the liver of D-gal-treated mice. Normal controls were treated
with saline (0.9%), and D-gal mice were treated with D-gal daily for 8 weeks.
FGF21-treated D-gal
mice
(D-gal+FGF21)
were treated
with
D-gal
and
simultaneously administrated with FGF21 sc at doses of 1 or 5 mg·kg-1·d-1 once a day
for 8 weeks. NAC-treated D-gal mice (D-gal+NAC) were treated with D-gal and
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simultaneously administrated with N-acetylcysteine (NAC) sc at doses of 5
mg·kg-1·d-1 once a day for 8 weeks. (A) ROS were measured with 2,
7-dichlorofluorescin-diacetate (DCFH-DA) fluorescence and determined using
Fluorescence Microplate Reader (Perkin Elmer, America). (B, C) MDA level and
SOD activity were measured by commercial detection kit using Microplate Reader
(Biotech Elx800). (D-F) CAT, GSH-Px and T-AOC activities were measured by
commercial detection kit using 722 spectrophotometer. All data are represented as
means ± SD, n = 8 per group. *P<0.05, **P<0.01 and ***P<0.001 vs D-gal group.
Fig. 2
B
1.5
***
1.0
0.5
**
*
0.0
Control
D-gal
CPT-1α mRNA expression
(relative level)
PGC-1α mRNA expression
(relative level)
A
5 mg/kg 1 mg/kg
1.5
**
**
1.0
**
0.5
0.0
Control
D-gal
D-gal+FGF21
D-gal+FGF21
C
D
2.0
Idh3α mRNA expression
(relative level)
SIRT1 mRNA expression
(relative level)
5 mg/kg 1 mg/kg
1.5
1.0
0.5
0.0
1.5
1.0
**
*
0.5
0.0
Control
D-gal
5 mg/kg 1 mg/kg
D-gal+FGF21
Control
D-gal
5 mg/kg 1 mg/kg
D-gal+FGF21
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F
1.5
1.0
***
0.5
**
*
0.0
Control
D-gal
5 mg/kg 1 mg/kg
D-gal+FGF21
NDUFAB1 mRNA expression
(relative level)
CytC mRNA expression
(relative level)
E
1.5
1.0
*
*
0.5
0.0
Control
D-gal
5 mg/kg 1 mg/kg
D-gal+FGF21
Cox5 mRNA expression
(relative level)
G
1.5
1.0
***
***
0.5
0.0
Control
D-gal
5 mg/kg 1 mg/kg
D-gal+FGF21
Supplemental Fig. 2. Effect of FGF21 on relative mRNA levels of mitochondrial
genes responsible for mitochondrial biogenesis and function in the liver of D-gal mice.
Normal controls were treated with saline (0.9%), and D-gal mice were treated with
D-gal daily for 8 weeks. FGF21-treated D-gal mice (D-gal+FGF21) were treated with
D-gal and simultaneously administrated with FGF21 sc at doses of 1 or 5 mg·kg-1·d-1
once a day for 8 weeks. (A) mRNA expression of PGC1-α. (B) mRNA expression of
CPT1α. (C) mRNA expression of SIRT1. (D) mRNA expression of Idh3α. (E) mRNA
expression of CytC. (F) mRNA expression of NDUFAB1. (G) mRNA expression of
Cox5. The mRNA expression was quantified by real-time PCR. Expression of each
gene relative to the expression of housekeeping gene, β-actin was analyzed and
calculated as 2−ΔΔCt. All data are represented as means ± SD, n = 8 per group. *P<0.05,
**P<0.01 and ***P<0.001 vs D-gal group.
Supplemental Materials and Methods:
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Animals and treatments
Eight-week-old male Kunming mice were purchased from Wei tong li hua
Animal Center (Beijing, China). The mice were maintained under constant conditions
(23 ± 1°C and 60% humidity) and had free access to rodent food and tap water. Eight
mice were housed per cage on a 12-h light/dark schedule (lights on 08:30–20:30). At
12 weeks of age (37.1±0.6g), mice were randomly divided into four groups (n=8 per
group), groups 2-5 received daily subcutaneous injection of D-gal (Sigma–Aldrich,
MO, USA) at dose of 180 mg·kg-1·d-1 for 8 weeks and group 1 as normal control with
injection of saline (0.9%) only. Meanwhile, group 3 and 4 D-gal-treated mice received
simultaneously sc FGF21 of 5 mg·kg-1·d-1 or 1 mg·kg-1·d-1, and group 5 D-gal-treated
mice receive simultaneously sc N-acetylcysteine (NAC) of 5 mg·kg-1·d-1. Then mice
were sacrificed and the livers were immediately collected, the ROS production, the
level of MDA and the activity of SOD, GSH-Px, CAT and T-AOC were measured by
commercial detection kit.
RNA isolation and real-time quantitative PCR
Total RNA from the livers was isolated with Trizol (Invitrogen), RNA was reverse
transcribed into cDNA using the reverse-transcription kit (Promega, USA). The cDNA
was used for real-time quantitative PCR (ABI 7500, Applied Biosystems, Carlsbad,
CA, USA) with SYBR Green Master Mix and melting curve to detect the following
genes: PGC1-α: (F: CAC CAA ACC CAC AGA AAA CAG; R: GGG TCA GAG
GAA GAG ATA AAG TTG). CPT1α: (F: AGA CAA GAA CCC CAA CAT CC; R:
CAA AGG TGT CAA ATG GGA AGG). SIRT1: (F: AGT TCC AGC CGT CTC TGT
GT; R: CTC CAC GAA CAG CTT CAC AA). IDH3α: (F: GAG GTT TTG CTG
GTG GTG TT; R: TCC TCC TGG TCC TTG AAT TG). CytC: (F: CCA AAT CTC
CAC GGT CTG TT; R: TAT CCT CTC CCC AGG TGA TG). NDUFAB1: (F: GGA
CCG AGT TCT GTA TGT CTT G; R: AAA CCC AAA TTC GTC TTC CAT G).
COX5b: (F: ACC CTA ATC TAG TCC CGT CC; R: CAG CCA AAA CCA GAT
GAC AG) and β-actin: (F: ACA TCT GCT GGA AGG TGG AC; R: GGT ACC ACC
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ATG TAC CCA GG). The amplified PCR products were quantified by measuring the
calculated cycle thresholds (Ct) of samples mRNA and β-actin mRNA. Relative
multiples of change in mRNA expression were calculated by 2−ΔΔCt. The mean value
of normal group target levels became the calibrator (one per sample) and the results
are expressed as the n-fold difference relative to normal controls (relative expression
levels).
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