A
B
Supplementary Figure S1. Monitoring of the body weight and ratios of liver to body weight in mice before and after RSV treatment. RSV (30 mg/kg/day) was orally administrated to the 4-week old (4-wk) WT and HBx TG mice once a day. The mice were sacrificed and livers were collected after RSV treatment at 2, 3, 7, and 14 days (d). There is no significant difference in the body weight (A) and ratio of liver/body weight (B) after RSV treatment at 2, 3, 7, and 14 days compared with the vehicle (H2O) group for the WT and HBx TG mice. Numbers in bars are mice. Bars represent mean
± SD.
0 5 10 15 20
25 Body weight (g)
WT HBx
H2O RSV
2 d WT HBx
(4-wk) H
2O RSV
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
0 d
3 3 4 4
4 3 8 8 8 7 9 8 5 8 5 6 3 6
0.01 0.02 0.03 0.04 0.05 0.06 0.07
0.08 Liver/body weight
0
WT HBx
H2O RSV
2 d WT HBx
(4-wk) H
2O RSV
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
0 d
3 3 4 4
4 3 8 8 8 7 9 8 5 8 5 6 3 6
Supplementary Figure S2. Serum ALT values before and after RSV treatment. RSV (30 mg/kg/day) was orally administrated to the 4-week old (4-wk) WT and HBx TG mice once a day. The mice were sacrificed and sera were collected at 2, 3, 7 and 14 days (d) after RSV treatment. Numbers in bars are mice. Bars represent mean ± SD. *p<0.05; **p<0.005.
Method: Measurement of serum alanine aminotransferase (ALT) level
Blood samples were obtained at various stages of RSV treatment. Serum ALT values were analyzed by DRI-CHEM 3500s (FUJIFILM).
Supplementary Figure S2
0 10 20 30 40 50 60
ALT (U/L)
** **
ALT (U/L)
WT HBx
H2O RSV
2 d WT HBx
(4-wk) H
2O RSV
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
0 d
4 3 3 3 3 3 8 7 5 5 4 5 3 8 4 5 3 5
B A
Scd1
Folds of mRNA level
0 0.2 0.4 0.6 0.8 1 1.2 1.4
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
3
3 3 3 3 3 3 3
3
3 3 3
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
- RSV RSV
4-wk 6-wk 12-mo 16-mo
Folds of mRNA level
- - -
HBx
3
3 3 3
3 3
C
Supplementary Figure S3_continued
Supplementary Figure S3. The mRNA levels of HBx, lipogenic genes Scd1, and LXR in the mouse livers with or without RSV treatment. (A) The mRNA levels of HBx were detected by real- time quantitative RT-PCR using total RNA isolated from the livers of HBx TG mice at 4-week old (4-wk), 6-wk, 12-month old (12-mo), and 16-mo (non-tumor part). There was a significant decrease of the HBx mRNA level in the pre-cancerous livers of HBx TG mice without any treatment; this had been reported and discussed previously (Wu et al., 2006; Lu et al., 2012). Nevertheless, expression of the HBx mRNA was not inhibited by RSV at the early stage of pathogenesis (RSV treatment from 4-wk to 6-wk for 14 days), or at a later pre-cancerous stage (RSV treatment from 12-mo to 16-mo for 4 months) in the HBx TG mice. (B) and (C) The mRNA levels of Scd1 and LXR were detected by real-time quantitative RT-PCR using total RNA isolated from the livers of WT and HBx TG mice.
RSV (30 mg/kg/day) was orally administrated to the 4-week old WT and HBx TG mice once a day.
The mice were sacrificed and livers were collected after RSV treatment at the indicated time points.
There was no significant difference in the mRNA levels of Scd1 and LXR in WT and HBx TG mice with or without RSV treatment. In (A) – (C), numbers in bars are mice. Bars represent mean ± SD.
References:
1. Wu BK, Li CC, Chen HJ, Chang JL, Jeng KS, Chou CK, et al. Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice. Biochem Biophys Res Commun 2006;340:916-928.
2. Lu JW, Hsia Y, Yang WY, Lin YI, Li CC, Tsai TF, et al. Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model.
Carcinogenesis 2012;33:209-219.
LXR
Folds of mRNA level
0 0.2 0.4 0.6 0.8 1.0 1.2
RSV RSV
WT HBx
H2O RSV
2 d H2O
3 d
H2O RSV H2O
WT HBx
0 d WT
- -
HBx
3 3 3
3 3 3 3 3 3 3
A
B
Akt
Gapdh p-Akt
Gapdh
2 d H2O RSV
WT
H2O RSV HBx
3 d H2O RSV
WT
H2O RSV HBx
Supplementary Figure S4. No significant difference in the ratios of p-Akt/Akt protein in the HBx TG mice with or without RSV treatment. RSV (30 mg/kg/day) was orally administrated to the 4-week old WT and HBx TG mice once a day. The mice were sacrificed and livers were collected after RSV treatment at the indicated time points. (A) A representative Western blot of the p-Akt and total Akt proteins in the livers of the WT and HBx TG mice after RSV treatment for 2 and 3 days (d). (B) Ratios of p-Akt/Akt after RSV treatment for 2 and 3 days. Numbers in bars are mice. Bars represent mean ± SD. *p<0.05.
0 0.1 0.2 0.3 0.4 0.5 0.6
H2O RSV H2O RSV
WT HBx
2 d
H2O RSV H2O RSV
WT HBx
3 d
*
Ratio of p-Akt / Akt
3 3 3
3 3 3 3 3
SirT1 activity
B
SirT1 protein
A
Supplementary Figure S5
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
0
Fold of SirT1 activity
0.5 1 1.5
2 2.5
3
* * * *
3 3 3
3 3 3 3 3 3 3 3 3
Fold of SirT1 protein level
0 0.5 1.0 1.5 2.0 2.5 3.0
3.5
* * * *
WT HBx
H2O RSV
3 d
H2O RSV
WT HBx
H2O RSV
7 d
H2O RSV
WT HBx
H2O RSV
14 d
H2O RSV
3 3 3
3 3 3 3 3 3 3 3 3
3 d
SirT1
-actin
RSV H2O
WT HBx WT HBx
7 d
14 d
SirT1
-actin
SirT1
-actin
C
Supplementary Figure S5. RSV stimulates the activity of SirT1 and increases SirT1 protein levels in the livers of the WT and HBx TG mice. RSV (30 mg/kg/day) was orally administrated to the 4-week old WT and HBx TG mice once a day. The mice were sacrificed and livers were collected after RSV treatment at the indicated time points. There is a significant increase in SirT1 activity (A) and SirT1 protein level (B) in both the WT and HBx TG mice after RSV treatment for 7 and 14 days (d). (C) Representative Western blot of the SirT1 protein after RSV treatment for 3, 7 and 14 days. The following antibody was used for the Western blot analysis: SirT1 (Sigma S5196, 1:1000). In (A) and (B), numbers in bars are mice. Bars represent mean ± SD. *p<0.05.
Method: Measurement of SirT1 activity
The activity of SirT1 was measured utilizing the SIRT1 Assay Kit (Sigma CS1040) according to the manufacturer’s instructions. The assay procedure is based on a two-step enzymatic reaction.
The first step is deacetylation by SIRT1 of a substrate that contains an acetylated lysine side chain.
The second step is the cleavage of the deacetylated substrate by the Developing Solution and the release of a highly fluorescent group. The measured fluorescence is directly proportional to the deacetylation activity of the enzyme in the sample. Liver tissues were homogenized in the
following buffer: 50mM Tris pH 7.5, 100mM NaCl, 1mM EDTA, 0.1% Triton X-100, 1mM NaF, 1mM Na3VO4, 1mM PMSF, protease inhibitor cocktail (Roche 11 836 153 001). We used 30 mg of proteins from each liver sample for the reaction.
A 12-mo HBx TG: Nodule 67% (6/9); HCC 0% (0/9) A-1: HBx 240 A-2: HBx 241 A-3: HBx CX057
A-4: HBx CX088 A-5: HBx CX089 A-6: HBx CX109
A-7: HBx CX164 A-8: HBx CX165 A-9: HBx CX166
Supplementary Figure S6
Supplementary Figure S6. Gross view of the livers in 12-month old and 16-month old HBx TG mice without treatment. (A) In the pre-cancerous mice at 12-month old (12-mo), hyperplastic nodules measuring between 0.5 to 2.5 mm in diameter could be detected in about 67% (6/9) of the HBx TG mice; however, there no HCC could be detected at this age. Arrows indicate hyperplastic nodules. (B) At 16-month old (16-mo), there was an 80% (8/10) incidence of HCC in the HBx TG mice without any treatment. All of the HCC samples were pathologically confirmed.
1cm
HCC
B
B-10: HBx 245
B-4: HBx 249 B-5: HBx 252
B-7: HBx CX91 B-8: HBx CX92
B-6: HBx 410
B-9: HBx D457
HCC
HCC
HCC HCC
HCC
Nodule
Nodule
B-2: HBx D480 B-1: HBx D476
HCC
B-3: HBx D481
1cmHCC
0 100 200 300 400 500
WT HBx WT HBx 12-mo
WT HBx WT HBx WT HBx
13-mo 14-mo 15-mo 16-mo
RSV - + - + - + - + - + - + - + - +
#507
#464
#508 P=0.29
ALT (U/L)
P=0.79 P=0.97
P=0.41
Supplementary Figure S7. Body weight (A) and serum ALT values (B) before and after RSV
treatment for the WT mice and the pre-cancerous stage of HBx TG mice from 12- to 16-month old. No obvious toxicity was detected in the WT mice after RSV (30 mg/kg/day) treatment for 4 months. RSV was supplemented in the chow (3 g/12.5 kg of chow) to provide a dose of approximately 30 mg/kg/day.
Serum ALT was monitored monthly. Mice were sacrificed and analyzed at 16-month old. All animals in this study were male mice. In (A), numbers in bars are mice. Bars represent mean ± SD.
B
A
Supplementary Figure S7
WT HBx WT HBx
12-mo 16-mo
RSV H2O RSV
H2O
- -
0 5 10 15 20 25 30 35 40 45 50
Body weight (g)
9 9 10 12 10 20
A
B
B-3: HBx D465 A-1: HBx D469
ALT (U/L)
12-mo (27)16-mo (14) Ventral
Dorsal
A-2: HBx D506
ALT (U/L)
12-mo (45)16-mo (33) Ventral
Dorsal
A-3: HBx D509
ALT (U/L)
12-mo (27)16-mo (23) Ventral
Dorsal
B-2: HBx D451 B-1: HBx D450
ALT (U/L)
12-mo (27)16-mo (14) Ventral
Dorsal
ALT (U/L)
12-mo (20)16-mo (20) Ventral
Dorsal
ALT (U/L)
12-mo (23)16-mo (37) Ventral
Dorsal
Group #2: Nodule size 0.5-2.5 mm, 55% (11/20)
1cm
1cm
B-4: HBx D466 B-5: HBx D467 B-6: HBx D493
B-9: HBx D497 B-8: HBx D495
B-7: HBx D494
ALT (U/L)
12-mo (31)16-mo (22) Ventral
Dorsal
ALT (U/L)
12-mo (21)16-mo (23) Ventral
Dorsal
ALT (U/L)
12-mo (31)16-mo (28) Ventral
Dorsal
ALT (U/L)
12-mo (28)16-mo (42) Ventral
Dorsal
ALT (U/L)
12-mo (30)16-mo (31) Ventral
Dorsal
ALT (U/L)
12-mo (33)16-mo (60) Ventral
Dorsal
B Group #2: Nodule size 0.5-2.5 mm, 55% (11/20)_continued
Supplementary Figure S8_continued
1cm
1cm
B-10: HBx D505 B-11: HBx D510
C Group #3: Nodule size 3-6 mm, 15% (3/20) C-2: HBx D496
C-1: HBx D468 C-3: HBx D498
ALT (U/L)
12-mo (24)16-mo (20) Ventral
Dorsal
ALT (U/L)
12-mo (29)16-mo (40) Ventral
Dorsal
ALT (U/L)
12-mo (22)16-mo (46) Ventral
Dorsal
ALT (U/L)
12-mo (37)16-mo (83) Ventral
Dorsal
ALT (U/L)
12-mo (21)16-mo (45) Ventral
Dorsal
B
1cm
1cm
D Group #4: HCC, 15% (3/20)
D-1: HBx D464 D-2: HBx D507 D-3: HBx D508
ALT (U/L)
12-mo (42)16-mo (470) Ventral
Dorsal
ALT (U/L)
12-mo (29)16-mo (452) Ventral
Dorsal
ALT (U/L)
12-mo (40)16-mo (110) Ventral
Dorsal
Supplementary Figure S8. Gross view of livers in the four groups of the HBx TG mice with RSV treatment for 4 months (from 12- to 16-month old). (A) Group #1, no grossly identifiable nodules;
there was a 15% (3/20) of mice in this group. (B) Group #2, livers contained small 0.5-2.5 mm hyperplastic nodules; there was a 55% (11/20) of mice in this group. (C) Group #3, livers contained 3-6 mm hyperplastic nodules; there was a 15% (3/20) of mice in this group. (D) Group #4, livers with HCC; there was a 15% (3/20) of mice in this group.
Supplementary Figure S8_continued
1cm
