1
Supplementary Fig. S1.
(a) Effects of ALK5 inhibitors on 3TP-Lux promoter activity induced by TGF-β1. HaCaT 3TPLux stable cells were treated with the indicated drugs for 24 h. The cells were analyzed by
luciferase assays. Mean luciferase activities are expressed as a percent of the control (n = 3). (b)
ALK5 mRNA and protein were silenced by transfection of ALK5 siRNA into LX-2 and
Hepa1c1c7 cells. After transfection, mRNA (left) and protein (right) expression of the ALK5
gene was evaluated. GAPDH was used as a reference.
transfected cells,
**
p<0.01 vs. nontargenting (NT)-siRNA
***
p<0.001 vs. NT-siRNA transfected cells. (c) Effects of EW-7197 on
phosphorylation of Smad1/5/8 in LX-2 and Hepa1c1c7 cells. The cells were treated with the
indicated drugs in the presence or absence of TGF-β1 for 3 h. Smad1/5/8 was used as a
reference. (d-g) Western blot analysis of p-Smad3 (upper) and Smad3 (lower) in CCl4 mice (d),
BDL rats (e), UUO mice (f), and BLM mice (g).
Supplementary Fig. S2.
(a-d) Western blot analysis of α-SMA in CCl4 mice (a), BDL rats (b), UUO mice (c), and BLM
mice (d).
Supplementary Fig. S3.
(a-d) Western blot analysis of COL-1 in CCl4 mice (a), BDL rats (b), UUO mice (c), and BLM
mice (d).
Supplementary Fig. S4.
(a-c) Western blot analysis of αv integrin (upper panel) and fibronectin (lower panel) in CCl4
mice (a), BDL rats (b), and UUO mice (c).
2
Supplementary Fig. S5.
(a) Western blot analysis of 4-HNE in liver tissues of CCl4 mice. (b) Densitometric analysis of
western blots of p38 phosphorylation in liver tissues of CCl4 mice. p38 was used as a reference.
***
p<0.001 vs. Sham, ##p<0.01 vs. CCl4.
Supplementary Fig. S6.
(a) Western blot analysis of NOX1 (upper) and NOX2/gp91phox (lower) in liver tissues of CCl4
mice. (b) Western blot analysis NOX4 in kidney tissues of UUO mice.
Supplementary Fig. S7.
(a) Western blot analysis of Prdxs in liver tissues of CCl4 mice. (b) Western blot analysis of
Prdx1 in liver tissues of CCl4 mice.
Supplementary Fig. S8.
(a-d) Western blot analysis of GAPDH in CCl4 mice (a), BDL rats (b), UUO mice (c), and BLM
mice (d).
3
4
5
6
7
8
9
10
11
12
13
14
Table S1. siRNA sequence
Sense
Antisense
#1
CUCGUACUUAUUGUCAGUA
UACUGACAAUAAGUACGAG
#2
GUCCGUUUCAUACGUCAGA
UCUGACGUAUGAAACGGAC
#3
GAGAAGAGCGUUCAUGGUU
AACCAUGAACGCUCUUCUC
#1
CUGACCCAUCAGUUGAAGA
UCUUCAACUGAUGGGUCAG
#2
CAGACUUAGGACUGGCAGU
ACUGCCAGUCCUAAGUCUG
#3
CGAUUUGGAGAAGUUUGGA
UCCAAACUUCUCCAAAUCG
Mouse ALK5
Human ALK5
15
Table S2. Antibodies for western bolt, immunofluorescence, and IHC.
Antibody
Company
Product number
4-HNE
Abcam
ab46545
α-SMA
Sigma
A2547
ALK5
Santa cruz biotechnology
sc-398
αv integrin
Cell signaling
4711
COL-1
Santa cruz biotechnology
sc-59772
Fibronectin
BD biosciences
610077
GAPDH
Cell signaling
5174
NOX1
Abcam
ab55831
NOX2/gp91phox
Santa cruz biotechnology
sc-5827
NOX4
Santa cruz biotechnology
sc-30141
Prdx
Supplied by Dr. HA Woo (Ewha Womans University, Seoul, Korea)
p-p38
Cell signaling
4511
p38
Cell signaling
8690
p-Smad3
Cell signaling
9520
Smad3
Ab frontier
AF9F7
p-Smad1/5/8
Cell signaling
9511
Smad1/5/8
Cell signaling
6944, 12534