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Figure
legends
FIGURE 1 PB inhibits the proliferation and activation of HSCs.
(A) The chemical structure of PB. (B-F) LX‐2 cells were treated with 2
ng·ml−1 TGFβ1 with or without different concentrations of PB for 24 hr
after no FBS starvation. (B) COL1A1 luciferase activity was determined
in LX-2 cells transfected with the
pGL4.17-COL1A1 luciferase construct.
(C) Cell proliferation was determined by CCK8 assay. (D)PB repressed theCOL1A1 , αSMA , TGFβ1 and TIMP1 in a
dose‐dependent manner in LX‐2 cells. GADPH served as loading
control. (E) PB dose‐dependently inhibited the protein levels of COL1A1
and α‐SMA in LX‐2 cells. The
protein levels were normalized against β-actin. (F) Immunofluorescence
staining for α-SMA (red) in LX-2 cells. (G-I) Mouse pHSCs were treated
with different concentrations of PB for 24 hr after 7 days of isolation.
(G) PB inhibited the col1a1 , and αsma in mouse pHSCs.β-actin served as loading control. (H) PB inhibited the protein
levels of α‐SMA in mouse pHSCs. The protein levels were normalized
against β-actin. (I) Immunofluorescence staining for α-SMA (red) in
mouse pHSCs. The values are expressed as the mean ± SEM of five
independent assays, *P < 0.05, **P < 0.01, ***P
< 0.001, ****P< 0.0001, significantly different from
the control group and #P < 0.05, ##P< 0.01,
###P< 0.001 significantly different from the TGFβ1
treatment group in LX‐2 cells and mouse pHSCs. ns, not significant.
FIGURE 2 PB markedly ameliorated the liver fibrosis in
CCl4‐treated mice. (A) Liver histopathology was observed
by H&E staining (magnification 200×). (B) Serum ALT, AST assays and (C)
Liver hydroxyproline concentration assays demonstrated that PB reduced
liver fibrosis in CCl4 mice.
(D)
The obtained liver sections were subjected to sirius red staining
(magnification 200×; up panel), and Masson trichrome stain
(magnification 200×; down panel). (E) mRNA expressions of αsma ,Col1a1 , Tgfb1 , and Timp1 in mice liver samples. (F)
Western blot analysis and semi‐ quantitation of α‐SMA expressions.
β-actin served as the loading control. The values are expressed as the
mean ± SEM (n = 8 of each group), *P < 0.05, **P <
0.01, ***P < 0.001, significantly different from control group
and ###P < 0.001, ####P < 0.0001,
significantly different from CCl4‐corn oil group. ns,
not significant.
FIGURE 3 PB markedly ameliorated the liver fibrosis in bile
duct‐ligated (BDL) mice. (A) Liver pathological changes were detected by
H&E staining (magnification 200×). (B) Serum ALT, AST assays and (C)
Liver hydroxyproline concentration assays demonstrated that PB reduced
liver fibrosis in BDL mice. (D) The degree of liver collagen
accumulation was determined by sirius red straining (magnification 200×;
up panel), and Masson trichrome stain (magnification 200×; down panel).
(E) mRNA expressions ofαsma , Col1a1 , Tgfb1 , and Timp1 in mice liver
sample. (F) Western blot analysis and semi‐quantitation of α‐SMA
expressions. β-actin served as the loading control. The values are
expressed as the mean ± SEM (n = 8 of each group), *P < 0.05,
**P < 0.01, ***P < 0.001, significantly different
from control group and ###P < 0.001, ####P <
0.0001, significantly different from
BDL group. ns, not significant.
FIGURE 4 PB suppressed the GLI1 expression in vitro andin vivo . (A) PB repressed GLI-luciferase activities. (B-C) PB
administration down‐regulated GLI1(B) mRNA and (C) protein expressions
in LX‐2 cells. GADPH /β-actin served as loading control. (D-E) PB
administration down‐regulated GLI1 mRNA(D) and (E) protein expressions
in mouse pHSCs. β-actin /β-actin served as loading control. (F) PB
administration down‐regulated GLI1 protein expressions in BDL livers.
β-actin served as loading control. The in vitro values are
expressed as the mean ± SEM of five independent assays, *P
<0.05, **P < 0.01, ***P < 0.001,
significantly different from the control group, #P < 0.05,
###P < 0.001significantly different from the TGFβ1
treatment group in LX‐2 cells. The in vivo values are expressed
as the mean ± SEM (n = 8 of each group), # P<0.05;
significantly different from sham group, *P<0.05;
significantly different from BDL-NS group. ns, not significant.
FIGURE 5 Altering GLI1 expression influenced the antifibrotic
activity of PB. (A-B) LX‐2 cells were transfected with 100 nM GLI1
siRNA1/2 or Ctrl siRNA (A) the mRNA expressions of fibrogenic gene were
analyzed by RT-qPCR. GADPH served as loading control. (B) Western blot
analysis and semi‐quantitation of CLI1, α‐SMA and Collagen α1(I)
expressions. β-actin served as the loading control. (C) LX‐2 cells were
transfected with 100 nM GLI1 siRNA1/2 or Ctrl siRNA and subsequently
treated with 2 ng·ml−1 TGFβ1 with or without different concentrations of
PB for 24 hr after no FBS starvation. The same protein expressions were
detected by western blot with the indicated antibodies. β-actin was used
as a loading control for all western blot assays. LX‐2 cells were
transfected with (D) 2.5‐μg HA‐GLI1 or vector for 6 hr and subsequently
treated with 2 ng·ml−1 TGFβ1 with or without different concentrations of
PB for 24 hr after no FBS starvation. The protein expressions of HA and
liver fibrosis markers were determined by immunoblotting with the
indicated antibodies. The data are expressed as the mean ± SEM of five
independent assays, *P <0.05, **P < 0.01, ***P
< 0.001, significantly different from the Ctrl siRNA group,
#P <0.05, ##P < 0.01, ###P < 0.001,
significantly different from the pcDNA /Ctrl siRNA group +TGFβ1 group,
▲P < 0.05, significantly different from the pcDNA + TGFβ1 + PB
1-μM group.
FIGURE 6 PB blocked the nuclear localization of GLI1 in HSCs.
(A) The nuclear and cytoplasmic extractions of LX‐2 cells were separated
after PB treatment and analysed by western blot. (B)LX‐2 cells and (C)
mouse pHSCs in dishes were fixed in 4% paraformaldehyde, stained with
DAPI (blue) and GLI1 (red), and imaged by confocal microscopy, Scale
bar: 20 μm. (D-E) The mRNA expressions of the GLI1 downstream genesHHIP , CYCLIN D , CYCLIN E and C-MYC (D) in
LX-2 cells and (E) in BDL group. The values are expressed as the mean ±
SEM of five independent assays, *P <0.05, **P <
0.01, ***P < 0.001, significantly different from the control
group, #P < 0.05, ###P < 0.001, significantly
different from the TGFβ1 treatment group. ns, not significant.
FIGURE 7 PB interrupted the
LAP2α/HDAC1
complex. (A) HA-GLI1 was transfected into HEK293T cells. Whole cell
extracts were immunoprecipitated
with
an anti-HA antibody. (B) Western blot analysis of
GLI1, LAP2α and HDAC1 expression
in LX-2 cells. β-actin served as a loading control. (C) Myc-LAP2α and
HA-GLI1 were transfected into HEK293T cells. The interaction between
Myc-LAP2α and HA-GLI1 was investigated using immunoprecipitation assays.
(D) Myc-LAP2α and FLAG-HDAC1 were transfected into HEK293T cells. The
interaction between Myc-LAP2α and FLAG-HDAC1 was investigated using
immunoprecipitation assays. The interaction between LAP2α and HDAC1 was
investigated using immunoprecipitation assays in LX‐2 cells (E) and (F)
CCl4-mice (G) LX‐2 cells in dishes were fixed in 4%
paraformaldehyde, stained with DAPI (blue), LAP2α (green) and HDAC1(red)
and imaged by confocal microscopy, Scale bar: 20 μm. The values are
expressed as the mean ± SEM of five independent assay.
FIGURE 8 LAP2α and HDAC1 were of significance to the
antifibrotic effects of PB. (A-C)
LX‐2
cells treated with PB (1 μM) and/or Vorinostat (10 μM, 6 hr) then (A)
the protein expressions of GLI1 and fibrogenic markers were detected by
immunoblotting with the indicated antibodies and (B) the protein
expressions of GLI1 were detected by immunofluorescence staining. (C)
HEK293T cells was transfected with HA-GLI1 and/or treated with PB (1 μM)
or Vorinostat (10 μM, 6 hr), Whole cell extracts were immunoprecipitated
with an anti-HA antibody. (D-G) 100‐nM LAP2α‐1/2 siRNA or Ctrl siRNA 48
hr. (D)The fibrotic mRNA and (E)protein expressions were detected by
western blot and RT-qPCR. (F) The protein expressions of GLI1 were
detected by immunofluorescence staining. (G) HEK293T cells was
transfected with HA-GLI1 and/or treated with PB (1 μM) or si‐LAP2α,
Whole cell extracts were immunoprecipitated with an anti-HA antibody.
The values are expressed as the mean ± SEM of five independent assays,
*P <0.05, **P < 0.01, ***P < 0.001,
significantly different from the control group.
FIGURE 9 Physalin B (PB) disrupted the interaction between
LAP2α and HDAC1, which enhanced the effect of GLI1
acetylation.
The increase of GLI1 acetylation further inhibited GLI1 exerting effect
on liver fibrosis.