1
SUPPLEMENTARY EXPERIMENTAL PROCEDURES
General methodology. Cell viability was determined by the MTT assay. Cell proliferation was
calculated from the rate of methyl[3H]-thymidine incorporation into the DNA of HSC. Details on
general methodology such as H&E staining, adenoviral infection, nuclear and cytosolic protein
isolation, Sirius red/fast green staining, measurement of ALT and Luc activities and gelatine
zymography to detect pro-, intermediate and active MMP2 and MMP9 in the cell culture medium
are described in previous publications from our laboratory (1-6).
Primary cell isolation. Primary HSC were isolated from male Sprague-Dawley rats (500 ± 25
g) by a 2-step in situ liver perfusion with pronase and liberase blendzyme-3 (Roche,
Indianapolis, IN). Hepatocytes and cellular debris were separated from non-parenchymal cells
centrifuging for 3 min at 50g. HSC were collected by density gradient centrifugation in 11% over
17.5% Histodenz. Cell purity (>95%) was assessed by cellular UV emission at 350 nm. Primary
HSC from male Opn-/- mice and their WT littermates (30 ± 3 g; n=3 mice/perfusion) were
isolated using collagenase IV (Sigma, St. Louis, MO). Primary human HSC were isolated from
normal liver margin of patients undergoing hepatic tumor resection for colorectal hepatic
metastases and were kindly donated by Dr. Hong (Mount Sinai School of Medicine, NY) (1-6).
Cell treatments. HSC (250,000 cells/well) were seeded on 6-well plates in DMEM/F12 with
10% FBS. Primary cells were cultured using DMEM-F12 for 4 to 7 days, which was replaced by
serum-deprived DMEM-F12 prior to endotoxin-free human rOPN treatment (1433-OP-050/CF,
R&D Systems, Minneapolis, MN). Time-course (5 min-48 hours) and dose-response (5-500 nM)
experiments were designed to determine the final concentration of rOPN (50 nM for rat and 100
nM for human HSC) and the best time-point for collagen-I induction (6-24 hours for rat and 1-24
hours for human HSC). Cells were infected with Ad-LacZ, Ad-OPN (7), Ad-Luc and Ad-NFκB-
2
Luc (8) at m.o.i. = 50 for 48 hours. The Ad-LacZ and Ad-OPN were donated by Dr. Uede
(Hokkaido University, Japan) and the Ad-Luc and Ad-NFκB-Luc were gifts from Dr. Engelhardt
(University of Iowa, IA). In the neutralization experiments, cells were incubated with 5 µg/ml of
non-immune IgG (Chemicon, Temecula, CA), anti-αvβ3 integrin (Chemicon), anti-CD44 (Lab
Vision, Fremont, CA), anti-OPN 2A1 (donated by Dr. Denhardt, Rutgers University, NJ) and
anti-TGFβ (R&D Systems) (Supplementary Table 1). The following treatments were added to
the cells: 0.1, 1 and 10 μM wortmannin (Calbiochem, San Diego, CA), 10 μM LY294002 (Cell
Signaling, Danvers, MA), 5 μM CAY10512 (Cayman Chemical, Ann Arbor, MI), 25 μM H2O2,
0.25 mM BSO, 2 mM glutathione-ethyl ester and 10 μM PDTC (all from Sigma).
HSC invasion. For the invasion assay, we used a modified transwell cell culture chamber. The
outer surface of an 8 μm-transwell was coated with rat collagen-I for 1 hour under sterile
conditions. HSC (25,000 cells/well) were seeded in serum-free medium on the upper chamber
and the lower chamber was filled with serum-free medium plus 0-50 nM rOPN and non-immune
IgG or neutralizing Abs to αvβ3 integrin and OPN. After 24 hours, the non-migrating HSC on the
upper surface of the filter were removed with a cotton swab and the cells that invaded the lower
side of the filter in the transwells were fixed in ice-cold methanol and stained with H&E. The
filters with fixed cells were detached from the transwells and mounted on glass slides. The
number of HSC present in 10 random fields at 100x were quantified as mean number of
migrating cells.
Wound healing in vitro assay. Rat HSC were seeded and grown to confluence after which a
mechanical wound was made on the center of the culture with a 200 μl pipette tip. Lifted cells
were removed using serum-free DMEM-F12 and 0-100 nM rOPN in the presence or absence of
non-immune IgG or neutralizing Abs to αvβ3 integrin or to OPN was added for 36 hours. A series
of multiple pictures of the wounds were captured at 200x using an inverted microscope.
3
Induction of liver injury. Opn-/- mice (B6.Cg-Spp1tm1Blh/J) and their matching WT littermates
(C57BL/6J) were obtained from Jackson Laboratories (Bar Harbor, Maine). A targeting vector
containing the neomycin resistance cassette and the Herpes simplex virus thymidine kinase
gene was used to disrupt exons 4 through 7 of the targeted gene. The targeted mutation deleted
the coding region of the Opn gene (9). The resulting chimeric animals were backcrossed to
C57BL/6J for ten generations. 129sv Opn-/- mice and their matching WT littermates were
generated and donated by Dr. Denhardt (Rutgers University, NJ) (10) and were used in
validation studies described in Supplementary Figures 5-6. In both cases, mice generated by
intercrossing Opn+/- mice and littermates were used in all experiments. The OpnHEP Tg mice
overexpressing OPN in hepatocytes with the OPN gene regulated under the serum amyloid P
component promoter along with their WT littermates were generated and donated by Dr.
Mochida (Saitama Medical University, Japan) (11). These mice were use in the experiments
described in Figure 8 and Supplementary Figure 7.
10-wks old male WT, Opn-/- and OpnHEP Tg littermates were used in this study. To induce acute
liver injury, mice received one i.p. injection of 0.5 ml/kg b.wt. CCl4 (Sigma) or equal volume of
MO and they were sacrificed 24 hours later. To induce chronic liver injury two in vivo models
were used. In the first model, mice were i.p. injected twice a week with 0.5 ml/kg b.wt. of CCl4 or
equal volume of MO for 1 month and they were sacrificed 48 hours after the last CCl4-injection.
In the second model, mice were treated with TAA (300 mg/L, Sigma) in the drinking water or
received equal volume of water for 2 or 4 months and they were sacrificed 48 hours after TAA
withdrawal. To determine the protective role of an antioxidant in vivo, WT mice were i.p. injected
with CCl4 or CCl4 plus SAM for 1 month. SAM was administered at a dose of 10 mg/kg b. wt.
daily and it was always given 2 hours before the CCl4-injection. Control groups received MO or
MO plus SAM.
4
All animals received humane care according to the criteria outlined in the ‘Guide for the Care
and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published
by the National Institutes of Health.
Human samples. Dr. Branch (Mount Sinai School of Medicine, NY) provided the human liver
protein lysates from de-identified controls and subjects with biopsy-proven stage-3 HCVcirrhosis. Samples were scored according to the Scheuer/Ludwig Batts classification. These
samples were IRB exempt since no patient information was disclosed.
Western blot analysis. Details on the Western blot methodology can be found in previous work
from our laboratory (1-6). Information on the source of commercially available Abs used for
Western blot analysis can be found in Supplementary Table 1. Anti-CYP2E1 Ab was a gift
from Dr. Lasker (Puracyp Inc., Carlsbad, CA) (12).
The 2A1 OPN Ab was generated and donated by Dr. Denhardt (Rutgers University, NJ) (13).
This Ab recognizes the epitope IPVAQ (13) and binds several OPN forms from mouse, rat and
human origin. The ~75 kDa species, when present, is the fully modified (glycosylated and
phosphorylated) monomeric OPN. The ~55 and ~25 kDa species are cleavage products of the
full-length OPN and a ~42 kDa cleavage product of unknown origin can be detected as well.
The smaller species may lack most or all of the post-translational modifications, or are more
likely cleavage products from thrombin, plasminogen, plasmin, cathepsin B and MMPs of the
full-length OPN (13) (Personal communication from Dr. Denhardt).
Anti-rat collagen-I was generated and provided by Dr. Schuppan (Harvard Medical School, MA).
The anti-rat collagen-I Ab detects procollagen α1(I) and α2(I) and N-terminally processed
5
pCα1(I) and pCα2(I), which run at ~165-200 kDa as well as collagen-I α1 and α2 chains, which
run at ~135 kDa. Human and mouse collagen-I were identified using an antibody from
Chemicon detecting mostly collagen-I α1 and α2 chains, which run at ~135 kDa. The
quantification under the blots refers to the sum of bands from all collagen-I isoforms in rat
samples and for the unique band in human and mouse samples. Intracellular collagen-I refers to
collagen-I detected in cells after removing the cell culture medium and washing with PBS twice;
thus, some collagen-I bound to the cells may remain. Extracellular collagen-I is the secreted
unbound collagen-I, precipitated with acetone (9:1, v/v) overnight and collected after
centrifugation at 11,000g for 30 min. Thus, total collagen-I refers to the quantification of intraplus extracellular collagen-I.
The ECL reaction was developed using the Las4000 scanner (Fujifilm, Stamford, CT). The
intensity of the Western blot bands was quantified using NIH ImageJ software. All Western blots
were carried out using triplicates and at least four different experiments. All samples from the
same experiment were run on the same gel and transferred onto the same nitrocellulose
membrane. All extracellular proteins analyzed by Western blot were corrected by total protein
content and protein loading was subsequently verified by Ponceau red staining on each
nitrocellulose membrane. In every experiment, the loading control used was β-tubulin, actin or
gapdh depending on the blot.
Pathology. In all experiments, the entire left liver lobe was collected and fixed in 10% neutralbuffered formalin and processed into paraffin sections for H&E or IHC staining. Inflammation
was noted to be lymphocytes present in the lobules and were scored as follows: 1 = rare foci, 2
= up to 5 foci and 3 when there were >5 foci. Ballooning degeneration was noted to be swelling
of hepatocytes with the cell membrane becoming prominent and rounded and the cytoplasm
was noted to be rarified. Often, clumps of cytoplasmic content were seen. A score of 1 was
6
given when rare cells were ballooned, a 2 when small groups of cells were ballooned and a
score of 3 when large groups were present, particularly when these were seen beyond the
centrilobular zones. Centrilobular necrosis and parenchymal necrosis were each separately
scored. The scores for centrilobular necrosis were 1 = hepatocyte necrosis affecting only zone
3, 2 = in addition to zone 3 necrosis, occasional bridging necrosis was seen, and 3 =
pronounced bridging and confluent necrosis. Parenchymal necrosis was noted to be spotty
necrosis or apoptosis in zones 2 and 1. The scores for parenchymal necrosis were 1 ≤1 focus, 2
= 5-10 foci, and 3 ≥10 foci at 100x. The degree of fibrosis ranged from 0 to 4 and patterned after
the Brunt system (14). Briefly, this was as follows: 1 = perisinusoidal/perivenular fibrosis alone,
2 = 1 plus portal fibrosis, 3 = bridging fibrosis and 4 = cirrhosis. In addition, the caliber of the
fibrosis in the sinusoids was further characterized as fine, thick and very thick. The assessment
of the above scores was uniformly performed in 10 fields/sample under 100x magnification
twice.
Immunohistochemistry. The 2A1 OPN Ab was used on IHC and on primary rat HSC. This Ab
was also tested in livers from Opn-/- mice under in C57BL/6J and 129sv genetic background to
ensure specificity (not shown) (13). The collagen-I Ab was from Chemicon and was used on
IHC. Reactions were developed using the Histostain Plus detection system (Invitrogen,
Carlsbad,
CA).
α-SMA-Cy3
(C6198,
mouse
IgG,
Sigma)
was
used
for
direct
immunofluorescence. The 2A1 OPN Ab (mouse IgG) was used on immunofluorescence
followed by an Alexa-488 conjugated goat anti-mouse IgG secondary Ab (Invitrogen). For the
Sirius red, OPN and collagen-I computer-assisted morphometry assessment, the integrated
optical density (IOD) was calculated from 10 random fields per section containing similar size
portal tract or hepatic vein at 100x and using Image-Pro 7.0 Software (Media Cybernetics,
Bethesda, MD). The results were averaged and expressed as fold-change over controls.
7
Statistical analysis. Data were analyzed by a two-factor ANOVA and results are expressed as
mean  SEM. All in vitro experiments were performed in triplicates at least four times. A
representative blot is shown in all figures. n=8/group mice per group were used in all the in vivo
experiments, which were repeated twice.
8
SUPPLEMENTARY LEGEND TO FIGURES
Supplementary Figure 1. Effects of rOPN on primary rat and human HSC viability and
proliferation rates. rOPN (0-50 nM) was added to rat HSC after a 24 hour starvation period at
4 and 7 days of culture and for 6 and 24 hours. Light micrographs showing similar rat HSC
phenotype and viability (A). Methyl[3H]thymidine incorporation showing only a slight increase in
rat HSC proliferation rates at 7 days when treated with 0-50 nM rOPN for 6 hours. Similar
results were obtained at 24 hours (not shown) (B). Light micrographs demonstrating equal
phenotype and viability in human HSC at 7 days of culture when incubated with 0-100 nM rOPN
for 6-24 hours (C). Methyl[3H]thymidine incorporation revealing increased human HSC
proliferation rates at 7 days when treated with 0-100 nM rOPN for 6 hours (not shown) or for 24
hours (D). Results are expressed as mean ± SEM. Experiments were performed in triplicates
four times. *p<0.05 for rOPN-treated vs control.
Supplementary Figure 2. rOPN induces rat HSC invasion and migration. Rat HSC were
seeded on transwells whose bottom side had been coated with rat collagen-I and cell
chemotaxis in the presence of 0-50 nM rOPN was determined 24 hours later by counting the
number of cells present on the bottom side of the transwells’ filter in 10 fields at 100x. Light
micrographs of H&E stained cells at 200x are shown in (A) and the number of HSC present in
10 random fields at 100x were quantified as mean number of migrating cells (B). Rat HSC were
seeded and 24 hours later, a mechanic wound was made on the plate with a 200 μl sterile tip.
Cells were incubated for 36 hours with 0-100 nM rOPN in the presence or absence of 5 μg/ml of
non-immune IgG or neutralizing Abs to αvβ3 integrin or to OPN and light micrographs were taken
(arrows point at highly proliferative cells) (C). Results are expressed as mean ± SEM.
Experiments were performed in triplicates four times. ***p<0.001 for rOPN-coated transwells vs
control.
9
Supplementary Figure 3. rOPN increases primary rat HSC collagen-I in a TGF independent manner. Incubation with 5 µg/ml of non-immune IgG or a TGFβ neutralizing Ab
did not prevent the collagen-I increase by treatment with 50 nM rOPN in rat HSC. Results are
expressed as mean ± SEM. Experiments were performed in triplicates four times. ***p<0.001 for
rOPN-treated vs control.
Supplementary Figure 4. The effects of rOPN on collagen-I in primary HSC involve
activation of the PI3K-pAkt signaling pathway. Incubation with 100 nM rOPN up-regulated
PI3K and the ratio pAkt
473
Ser/Akt up to 1 hour in human HSC (A). Primary rat HSC challenged
with 50 nM rOPN alone or in combination with 0-10 μM wortmannin did not show changes in cell
viability at 6 hours of treatment as shown by light micrographs (B). The rOPN-mediated
induction of collagen-I in human HSC was blunted by 10 μM wortmannin (C). Results are
expressed as mean ± SEM. Experiments were performed in triplicates four times. **p<0.01 and
***p<0.001 for rOPN-treated vs control. p<0.05, p<0.01 and p<0.001 for co-treated or
wortmannin-treated vs rOPN-treated or control.
Supplementary Figure 5. WT mice in 129sv genetic background show more CCl4-induced
chronic liver injury than Opn-/- mice. 129sv WT and Opn-/- mice were injected CCl4 or MO for
1 month. H&E staining showed more centrilobular necrosis () and inflammation () in CCl4injected WT than in Opn-/- mice (A). ALT activity (B), centrilobular and parenchymal
inflammation (C) and centrilobular and parenchymal necrosis (D). A Western blot analysis
shows similar CYP2E1 expression in WT and Opn-/- mice (E). Results are expressed as mean
values ± SEM. n=8/group; ***p<0.001 for CCl4 vs MO; p<0.05, p<0.01 and p<0.001 for
Opn-/- + CCl4 vs WT + CCl4.
10
Supplementary Figure 6. WT mice in 129sv genetic background show more CCl4-induced
fibrosis than Opn-/- mice. 129sv WT and Opn-/- mice were injected CCl4 or MO for 1 month.
Sirius red/fast green staining indicated fibrosis stage <3 in CCl4-injected WT and stage ~1-2 in
CCl4-injected Opn-/- mice as well as notable portal () and bridging () fibrosis (A). IHC for
collagen-I showed portal fibrosis (), bridging fibrosis () and scar thickness () in CCl4injected WT mice (B). The Brunt fibrosis score is shown in (C), the Sirius red and the collagen-I
morphometry analysis are shown in (D-E). Results are expressed as mean values ± SEM.
n=8/group; ***p<0.001 for CCl4 vs MO; p<0.01 and p<0.001 for Opn-/- + CCl4 vs WT + CCl4.
Supplementary Figure 7. OpnHEP Tg mice in C57BL/6J genetic background develop
spontaneous fibrosis. WT and OpnHEP Tg mice were maintained under normal chow diet in the
absence of a profibrogenic treatment for 1 yr, after which mice were sacrificed. Sirius red/fast
green staining, collagen-I IHC and morphometry analysis show perivenular, perisinusoidal and
portal fibrosis in OpnHEP Tg compared to WT mice. Results are expressed as mean values ±
SEM. n=3/group; **p<0.01 for OpnHEP Tg vs WT.
Supplementary Figure 8. WT show more TAA-induced fibrosis than Opn-/- mice. C57BL/6J
WT and Opn-/- mice received TAA or water for 4 months. Sirius red/fast green staining showing
greater collagenous proteins in TAA-treated WT compared to Opn-/- mice (A). IHC depicted
more collagen-I in TAA-treated WT than in Opn-/- mice (B). In (A) and (B) scar thickness (),
portal fibrosis () and bridging fibrosis () were higher in TAA-treated WT than in Opn-/- mice.
The Brunt fibrosis score shows stage >3 in TAA-treated WT and ~1-2 in Opn-/- mice (C). Sirius
red and collagen-I morphometry assessment demonstrated similar results (D-E). Results are
11
expressed as mean values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for TAA vs water;
p<0.05 and p<0.001 for Opn-/- + TAA vs WT + TAA.
Supplementary Figure 9. Proposed mechanism for the effects of rOPN on collagen-I
regulation and liver fibrosis. Chronic liver injury and oxidant stress induce OPN expression in
HSC. OPN engages αvβ3 integrin, increases PI3K and induces the ratio pAkt
473
Ser/Akt. These
occur along with activation of the NFκB signaling pathway as shown by the increased ratios of
pIKKα,β
Ser/IKKα,β and pIκBα
176/180
Ser/IκBα as well as by nuclear translocation of p65.
32
These events lead to up-regulation of intra- and extracellular collagen-I protein. These rOPNmediated effects are prevented by a neutralizing Ab to integrin αvβ3, and by inhibitors of PI3K
activation (wortmannin and LY294002) and NFκB signaling (PDTC and CAY10512). CD44
engagement, the mTOR/p70S6K signaling pathway and other oxidant stress-activated kinases
(i.e. pp38, pERK1/2 and pJNK) do not play a major role in these effects. Moreover, rOPN
induces HSC invasion and migration and lowers MMP13 expression; thus, favoring scarring.
Taken as a whole, all these factors regulating collagen-I protein expression in HSC under rOPN
treatment contribute to the development of scarring and liver fibrosis.
12
Supplementary Table 1. List of commercially available antibodies used.
Target
Ab
Source
Actin
sc-1616
Santa Cruz Biotechnology
Collagen-I
MAB3391
Chemicon
GAPDH
sc-20357
Santa Cruz Biotechnology
IKKα,β
2682
Cell Signaling
IκBα
4814
Cell Signaling
MMP1
AB19140
Chemicon
MMP13
MAB13426
Chemicon
mTOR
2983
Cell Signaling
p65
4764
Cell Signaling
P70S6K
sc-230
Santa Cruz Biotechnology
PI3K
sc-7189
Santa Cruz Biotechnology
p-IKKα,β 176/180Ser
2697
Cell Signaling
32
p-IκBα Ser
2859
Cell Signaling
p-mTOR 2448Ser
2971
Cell Signaling
2481
p-mTOR
Ser
2974
Cell Signaling
α-SMA
A2547
Sigma
β-Tubulin
T4026
Sigma
αVβ3 Integrin
MAB1976
Chemicon
CD44
MS178P1ABX Lab Vision
TGFβ
AB-100-NA
R&D Systems
Akt1/2/3
sc-1618
Santa Cruz Biotechnology
423
p-Akt1/2/3 Ser
sc-7985-R
Santa Cruz Biotechnology
13
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