Supplementary Data - Journal of Hepatology

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Supplementary Materials and methods
Biochemical Assays
Post-sacrifice, mouse serum was assessed for alkaline phosphatase (ALP),
aspartate transaminase (AST) and alanine transaminase (ALT) levels (U/L) by
Sydney South West Pathology Service.
Quantitative RNA analyses
RNA from frozen liver tissue was extracted using TRIzol (Invitrogen), cDNA was
generated from DNase I-treated RNA (1μg) using SuperScript III (Invitrogen),
and gene expression levels were determined using TaqMan assays (Applied
Biosystems, Suppl. Table 1) as per supplier instruction. Cycling conditions
included 40 cycles of initial denature (95oC, 10min); denature/extension (95oC,
15s; 60oC, 1min). Data are expressed as either copy of the gene of interest per
copy of 18S rRNA (standard curve method), or relative to 18S rRNA levels, as
indicated.
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Supplementary Table 1. TaqMan mouse gene expression assays.
Primer/Probe
Set
18S
Eukaryotic 18S ribosomal RNA
Inventoried Assay
ID
4352930E
Acta2
Actin, alpha 2, smooth muscle,
Mm01546133_m1
Bcl2
B cell leukemia/lymphoma 2
Mm00477631_m1
Cdh1
Cadherin 1 (aka E-cad)
Mm01247357_m1
Col1a1
Collagen, type I, alpha 1
Mm00801666_g1
Gli1
GLI-Kruppel family member GLI1
Mm00494654_m1
Gli3
GLI-Kruppel family member GLI3
Mm00492333_m1
Hhip
Hedgehog interacting protein
Mm00469580_m1
Ifng
Interferon gamma
Mm00801778_m1
Il-6
Interleukin 6
Mm00446191_m1
Ptc1
Patched homolog 1
Mm01306905_m1
Shh
Sonic hedgehog
Mm00436528_m1
Snai1
Snail homolog 1 (Drosophila)
Mm00441533_g1
Tgfb1
Transforming growth factor, beta 1
Mm03024053_m1
Tnf
Tumor necrosis factor
Mm00443258_m1
Vim
Vimentin
Mm01333430_m1
Zeb2
Zinc finger E-box binding homeobox 2 Mm00497193_m1
Gene Name
Supplementary Table 2. Primers for in situ hybridisation PCR template
generation including T7 or T3 RNA polymerase promoter sequences.
Target
Type
Primer Sequence (5’  3’)
mShh
Forward;
T7
Reverse;
T3
Forward;
T7
Reverse;
T3
TAATACGACTCACTATAGGGAGAGGGTTTGG
AAAGAGGCGGCACCC
ATTAACCCTCACTAAAGGGAGACACGGAGTT
CTCTGCTTTCACAG
TAATACGACTCACTATAGGGAGACCCAGCTC
GCTCCGCAAACA
ATTAACCCTCACTAAAGGGAGAGGTGCGCCA
GCGTGGGTTA
mShh
mGli1
mGli1
Product
Size (bp)
489
455
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Histopathology and immunohistochemistry
Mouse 5μm paraffin liver sections were used for H+E, Picrosirius red (PSR)
and immunohistochemistry. H+E and PSR staining was carried out by staff from
the Histopathology Lab, University of Sydney. Heat-mediated antigen retrieval
was performed using Universal Decloaker (Biocare Medical). Endogenous
peroxidase activity was blocked with 3% (v/v) hydrogen peroxide in methanol.
Primary antibodies to detect α-SMA (ab32575), CK7 (ab90083) and IHH (sc13088) were used. Following HRP-conjugated secondary antibody addition
(Dako), signal was detected using DAB. Isotype IgG controls and secondary
antibody alone controls returned no specific staining (Suppl. Fig 1B).
Immunofluorescence
Frozen liver sections (4μm) were fixed in 10% neutral buffered formalin or 1:1
acetone/methanol. Sections were incubated in primary antibody (Suppl. Table
3), prior to addition of an AlexaFluor-conjugated secondary (Invitrogen). Mouse
primary antibodies were directly conjugated to CF dyes using Mix-n-StainTM
CFTM Dye Antibody Labelling kit (Biotium). Isotype IgG controls were conducted
in parallel (Suppl. Fig. 1A,C).
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Supplementary Table 3. Primary antibodies used for immunofluorescence.
Marker
Protein
Catalogue ID
Supplier
α-ac-tubulin
α-acetylated tubulin
T6074
Sigma-Aldrich
γ-tubulin
γ-tubulin
C7604
Sigma-Aldrich
CD45
Leukocyte common antigen
550539
BD Pharmingen
CK18
M7010
Dako
sc-53532
Santa Cruz
Z0334
Dako
ab7181 ab7195
Abcam
Pan-CK
Cytokeratin 18
Epithelial cell adhesion
molecule
Glial fibrillary acidic protein
GLI- Kruppel family member
GLI2
Pan-cytokeratin
Z0622
Dako
Pan-Hh
Hedgehog ligand (SHH, IHH)
sc-1194
Santa Cruz
SHH
Sonic Hedgehog
ab53281
Abcam
SMO
Smoothened
LS-A2668
LifeSpan
Vimentin
Vimentin
MAB2105
R&D
EpCAM
GFAP
GLI2
Western blot
Whole liver tissue was homogenised in RIPA buffer containing protease and
phosphatase inhibitors (Roche). Protein lysates (10-60μg) were separated by
SDS-PAGE and transferred to PVDF. Membranes were incubated in primary
antibody overnight (Suppl. Table 4) and detected using ECL HRP substrate.
GAPDH demonstrated equal protein loading of whole liver and cell lysates.
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Supplementary Table 4. Primary antibodies used for Western blot.
Marker
Protein
Catalogue ID
Supplier
α-SMA
α-smooth muscle actin
BCL2
B-cell lymphoma 2
CYCLIN D1
Cyclin D1
Epithelial cell adhesion
molecule
Glyceraldehyde-3-phosphate
dehydrogenase
GLI-Kruppel family member
GLI1
GLI-Kruppel family member
GLI3
Hepatocyte nuclear factor 3β
ab32575
554218
#2876
#2926
Abcam
BD Pharmingen
Cell Signalling
Cell Signalling
ab32392
Abcam
AM4300
Ambion
sc-20687
Santa Cruz
sc-6155
Santa Cruz
#3143
Cell Signalling
sc-21742
Santa Cruz
sc-9024
Santa Cruz
PTCH1
Osteopontin
Hedgehog ligand (SHH, IHH,
DHH)
Patched 1
ab5315
Abcam
SMO
Smoothened
ab72130
Abcam
V5
V5-tag
R960-25
Invitrogen
EpCAM
GAPDH
GLI1
GLI3-R
HNF3β
OPN
Pan-Hh
Cell culture
Murine liver hepatocyte cell line H2.35 (CRL-1995™) was sourced from
CellBank Australia (CRMI, Westmead, Australia) and cultured per distributor’s
instructions. The murine LPC line BMOL1.2 and human hepatic stellate cell line
LX2 were propagated as previously described[1, 2]. BMOL1.2 media did not
include EGF. Where appropriate, cells were selected using blasticidin (5μg/mL)
for 7 days.
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Vector Construction
Human GLI1 (hGLI1) and GLI3-repressor (hGLI3R) cDNAs were subcloned
into the pDONR221 gateway donor vector (Invitrogen). Recombination into
pEF-DEST51 was performed as per manufacturer’s instructions. DNA
containing eight concatenated GLI transcriptional response elements (8xGli
minimal response promoter) was cloned into the promoterless luciferase
reporter pGL4.10[luc2] (Promega, USA).
Primary cilia (Pc) quantitation
Double immunofluorescent staining to detect Pc (α-acetylated tubulin) and LPC
marker EpCAM was performed in frozen mouse liver sections (WT TAA-20wk),
as described previously. To quantify the number of Pc+/EpCAM+ vs. Pc/EpCAM+ cells, ten random fields of view (located within the fibrous septa) were
counted/section (n=4), using the 100x objective. The number of Pc+/EpCAM+
cells and total number of EpCAM+ cells were recorded per field of view.
Statistics
All data are expressed as mean + standard error of the mean (S.E.M). A p<0.05
was considered significant. Statistical analyses were performed as described
within the text or Figure legend. For Fig. 2A, data were analyzed using Multiple
pairwise comparisons, One-way ANOVA, Bonferroni corrected. Significance
difference between means (*) are indicated with p<0.0057 for Gli1, p<0.0038
for Gli3, and p<0.0013 for Hhip. Alternatively, a One-sided student t-test was
used in analysis of Ptc1, *p<0.05.
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Supplementary References
[1]
Tirnitz-Parker JE, Tonkin JN, Knight B, Olynyk JK, Yeoh GC. Isolation, culture
and immortalisation of hepatic oval cells from adult mice fed a choline-deficient,
ethionine-supplemented diet. Int J Biochem Cell Biol 2007;39:2226-2239.
[2]
Xu L, Hui AY, Albanis E, Arthur MJ, O'Byrne SM, Blaner WS, et al. Human
hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut
2005;54:142-151.
Supplementary Figure legends
Supplementary. Fig. 1. IgG controls for immunofluorescence and
immunohistochemistry. (A) As a negative control for immunofluorescence
studies, goat IgG (Hh ligand sc-1194), rabbit IgG (GLI2 ab7181) or rat IgG2a
(vimentin MAB2105) was substituted at the same final concentration as the
primary antibodies listed. 20x objective, TAA-20wks frozen liver tissue. (B)
Representative rabbit IgG negative control for immunohistochemistry (IHH, αSMA or cytokeratin 7) using paraffin embedded TAA-20wks liver tissue. 20x
objective. (C) IgG controls for TAA 20wk/human ALD confocal microscopy
studies, 63x objective. DAPI, blue. No specific staining was observed. GLI2 IgG
controls: rabbit IgG/rat IgG controls.
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Supplementary Fig. 2. Liver histology and collagen deposition in the TAA
model. Mouse paraffin liver sections from control 20wks and TAA-treated (4,
8, 20wks) mice were stained for (A) haemotoxylin and eosin (H+E). 20x
objective. (B) Picrosirius red (PSR) staining. PSR detects collagen I (red),
indicative of the degree of fibrosis. 10x objective.
Supplementary Fig. 3. Liver injury markers in the TAA model. Mouse serum
or whole liver samples were obtained from control 20wk and TAA-treated (4, 8,
20wk) mice. (A) Quantitation of ALP, ALT and AST enzyme levels (U/L) in
mouse serum. Plots represent mean+S.E.M (n = 3-10 mice/group). Asterisk
denote significant difference between means (TAA-treated group vs. control
20wk, or between treatment groups as indicated). Multiple pairwise
comparisons, One-way ANOVA, Bonferroni corrected, ****p<0.0001. (B) qRTPCR to detect col1-1a, Acta2, Tgfb1, Tnf, Il-6 and Ifng transcripts. Data
expressed as copies of the gene of interest per copy of 18S rRNA. Mean+S.E.M
(n = 3-6 mice/group). Kruskal-Wallis followed by Dunn’s multiple comparisons
test; *p<0.05, **p<0.01 vs. control 20wk.
Supplementary Fig. 4. Expansion of hepatic stellate cell and liver
progenitor
cell
populations
following
TAA
treatment.
(A)
Immunohistochemistry to detect α-SMA (brown), expressed by activated HSCs
or immunofluorescence to detect EpCAM (red), expressed by LPCs. 10x
objective (top row), 20x objective (bottom row). (B) Immunohistochemistry to
detect CK7+ve LPCs, present at the epithelial-mesenchymal interface. 40x
objective.
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Supplementary Fig. 5. Hh ligand protein was not detected in leukocytes
or hepatic stellate cells in vivo, although small portal tract cells were
Shh+ve. (A) High power view of Shh mRNA detected by ISH, with Shh+ve cells
located at the epithelial-mesenchymal interface. Shh+ve hepatocytes (dashed
white arrows). Shh+ve small cells (solid white arrows). 40x objective. (B) Double
immunofluorescence determined that Hh ligand (SHH, IHH; red) proteins were
not expressed by CD45+ve leukocytes (green), or glial fibrillary acidic protein
(GFAP+ve)/vimentin+ve
activated
HSCs/myofibroblasts
(green)
Confocal
microscopy, 63x objective.
Supplementary Fig. 6. SHH is expressed by hepatocytes in human
alcoholic liver cirrhosis. Immunofluorescence was performed using human
ALD tissue to detect SHH and hepatocyte marker CK18. (A) Low power view
of SHH (red) throughout the hepatic nodule. (B) High power view demonstrates
SHH expression restricted to hepatocytes at the periphery of the nodule, and
lack of expression within small cells through the fibrous septa. (C) Vesicular
SHH staining within CK18+ve hepatocytes. Confocal microscopy, 63x objective.
Supplementary Fig. 7. Co-localisation of GLI2 and Hh ligands within
hepatocytes. Double immunofluorescence using frozen TAA-20wks tissue
identified what cell populations co-expressed GLI2 and pan-Hh (SHH, IHH). (A)
Image obtained at the epithelial-mesenchymal interface. Pan-Hh (red), GLI2
(green). (B) Demonstration of pan-Hh and GLI2 co-localisation within
hepatocytes. Confocal microscopy, 63x objective.
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Supplementary Fig 8. Primary cilia are expressed by EpCAM+ve but not
vimentin+ve
cells
in
Ptc+/-
TAA-treated
mice
in
vivo.
Triple
immunofluorescent staining on frozen liver tissue from Ptc+/- TAA-8wk mice
identified what cell populations express primary cilia (Pc). (A) Pc (γ-tubulin, red;
α-acetylated tubulin, green) structures are expressed by EpCAM+ve (white)
LPCs (merged image, right). (B) Pc structures were not evident on vimentin+
(white) HSCs (left and right panels). Confocal microscopy, 63x objective.
Supplementary Fig. 9. Quantitation of primary cilia expression by LPCs;
Pc were not expressed by vimentin+ve HSCs during chronic liver injury in
vivo. (A) Quantification of Pc+/EpCAM+ vs. Pc-/EpCAM+ cells in TAA 20wk liver
tissue per field of view, 100x objective. (B) Pc structures (γ-tubulin, red; αacetylated tubulin, green) were not expressed by vimentin+ve (white) HSCs (left
and right panels). TAA-20wk liver tissue. Confocal microscopy, 63x objective.
Supplementary Fig. 10. EpCAM+ve liver progenitor cells (LPCs) are in
close association with activated hepatic stellate cells, and express
several LPC markers. Double immunofluorescence to detect (A) EpCAM+ve
(green) LPCs in relation to α-SMA+ve activated HSCs. Co-localisation (yellow)
of EpCAM (green) with LPC markers (B) CK19 (red), and (C) CD133 (red). 40x
objective.
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Supplementary Fig. 11. Primary cilia expression in liver cell lines.
Immunofluorescence was performed to detect what liver cell lines express
primary cilia (Pc) (γ-tubulin, red; α-acetylated tubulin, green). (A) Pc were
detected on the murine progenitor cell line, BMOL1.2. Scale bars 12 μm (left),
3.9 μm (inset). Pc were not detected on (B) murine hepatocyte cell line, H2.35
or (C) human hepatic stellate cell line, LX2.
Supplementary Fig. 12. Quantification of EMT-related genes in Ptc+/- vs.
wt TAA-treated mice and correlation with Gli1. (A) qRT-PCR for EMTrelated genes E-cadherin (Cdh1) and vimentin (Vim) mRNA in whole liver. (B)
qRT-PCR for EMT-related Hh-target genes Bcl2, Snai1 and Zeb2 mRNA in
whole liver. (A, B) Mean+S.E.M., Mann-Whitney U test; *p <0.05, **p <0.01. (C)
Significant correlation of Gli1 mRNA (Hh activation) with EMT-related Hh-target
genes Bcl2 (Gli1 vs. Bcl2; Spearman r = 0.7053, p = 0.0005), Snai1 (Gli1 vs.
Snai1; Spearman r = 0.6264, p = 0.0054) and Zeb2 (Gli1 vs. Zeb2; Spearman
r = 0.7213, p = 0.0003) mRNA.

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