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Supplementary materials and methods.
Mouse hepatocyte isolation and culture.
Primary hepatocytes were isolated from livers of male Black6 mice (8-12 weeks of age)
using collagenase perfusion as described previously (1). Cells were seeded on plates precoated with a solution of 250 μg/mL of rat tail collagen I for monolayer cultures (CM), or
between two layers of collagen gel referred to as Collagen Sandwich (CS). For CS, a
gelled matrix was obtained by neutralization of a 0.75 mg/ml rat tail collagen I solution,
followed by incubation at 37 °C for 30-60 minutes. For both culture conditions, the cells
were plated at a density of 4x10 4 cells/cm2 in Williams' medium E (2 mM L-glutamine, 1
% penicillin/streptomycin) plus 10% FBS and 100 nM dexamethasone. 4h after
attachment, the cells were washed 3 times with HBBS, and incubated over night with
serum free Williams’ medium E (2 mM L-glutamine and 1 % penicillin/streptomycin) plus
100 nM Dexamethasone. For CS, after washing, a second layer of collagen gel was added
and allowed to polymerize for 30 min at 37°C, then added William’s medium like in CM
culture. After over night serum starvation, control cells were kept in Williams’ E plus P/S
and L-Glutamine. Stimulations were done in serum free medium for both culture
conditions. In some experiments, hepatocytes were seeded onto CM followed by addition
of an upper layer of collagen gel (CM+CG), or simply plated onto a single layer of
collagen gel (CG). Collagen preparation and medium changes were performed as
described for CS.
Cell lines. Non-differentiated AML12 mouse hepatoma cells were grown in
F-12/DMEM
complete medium (Invitrogen, Karlsruhe, Germany) as described (2). Cells were plated
at a density of 3x105 or 20x104 cells/well in 6-well plates or 24 well plates respectively,
and starved over night in 0% FBS medium before TGF-β stimulation.
Immunofluorescence and confocal microscopy. Immunostainings were performed
as previously described (2). Briefly, primary hepatocytes ( in CM or CS) or and AML12
cells, were fixed in 4% PFA/PBS for 10 minutes, permeabilized with 0,3% Triton X-100
in PBS for 15 minutes and washed 3 times in PBS. For staining of F-actin fibers, the cells
were incubated with 500 ng/mL phalloidin-rhodamin in PBS for 1h at room temperature.
For E-cadherin immunostaining, fixed and permeabilized cells were blocked in 5%
BSA/PBS for 1h, then incubated with mouse anti-E-Cadherin for 1h at room temperature,
washed 3 times in PBS, then incubated with Alexa-555-goat anti–mouse IgG (Molecular
Probes/Invitrogen).
Nuclei
were
stained
with
SYTOX®
Green
(Molecular
Probes/Invitrogen) for AML12 cells or DRAQ5® (Biostatus Ltd. Shepshed Leicesteshire,
UK) for primary hepatocytes. Samples were mounted using DakoCytomation Fluorescent
Mounting Medium (DakoCytomation, Hamburg, Germany). Confocal microscopy was
performed using a Leica laser scanning spectral confocal microscope, model DM IRE2,
with an HCX PL Apo 40 x/1.32 numeric aperture oil objective (Leica Microsystems,
Wetzlar, Germany). Excitation was performed with an argon laser emitting at 488 nm, a
krypton laser emitting at 568 nm, and a helium/neon laser emitting at 633 nm. Images
were acquired using a TCS SP2 scanner and Leica Confocal software, version 2.5 (Leica
Microsystems).
mRNA isolation, cDNA synthesis and PCR. Total mRNA was extracted from primary
mouse hepatocytes using the RNAeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was
synthesized from 1 µg RNA with the Quantitech Reverse Transcription kit (Qiagen,
Hilden, Germany). PCR was performed with Taq DNA Polymerase and dNTPack system
(Roche, Mannheim, Germany). The primers used are listed on Suppl. Table 2. PCR
products were resolved in 1% Agarose gels, and visualized by Ethidium Bromide staining.
Oligonuceotide microarray analysis
A total of 3 independent hepatocyte preparations were cultivated in collagen monolayer
for 1 and 24 h. Total RNA was collected from untreated or TGF- stimulated cells at each
time point and purified with the RNeasy Mini kit (Qiagen, Hilden, Germany), and the
integrity was verified by denaturating agarose electrophoresis. 5 µg RNA were
transcribed into cDNA by oligo dT primers, and reverse transcribed to biotinylated cRNA
with the Gene Chip IVT Labeling kit (Affymetrix, High Wycombe, UK). Hybridization to
arrays of type moe430_2 from Affymetrix (Santa Clara, CA, USA) and array scanning
were performed according to the recommendations of the manufacturer. Differential
expression
analysis
was
performed
with
ANOVA
in
BioConductor
(http://www.bioconductor.org) using R V2.4.1 (3). Raw fluorescence intensity values
were normalized applying quantile normalization. Gene expression values were converted
to a log2 scale. Significant differences in gene expression were analysed with limma
package for R Multitest with False Discovery Rate. Genes with mean expression changes
greater than 2 fold (Log base 2 greater than 1/-1) and the p-value <0.05 were selected
as significantly changed. CustomCDF with Entrez based gene/transcript definitions R
package was used for gene annotation [Ferrari, 2007 #114]. Gene Ontology analysis was
done with DAVID (Database for Annotation, Visualization, and Interpreted Discovery;
http://apps1.niaid.nih.gov/david/, (4). GeneChips microarray study followed MIAME
guidelines issued by the Microarray Gene Expression Data group. We have deposited all
files
containing
the
raw
data
of
the
microarray
analysis
to
ArrayExpress
(http://www.ebi.ac.uk/arrayexpress/) at the European Bioinformatics Institute (Hinxton).
The data are available under accession number E-MEXP-1176.
SDS/PAGE Silver staining. To confirm equal yield of protein lysates on both CM and CS
cultures, we compared total protein lysates from both culture systems, obtained using
RIPA buffer and shear force with a 20 gauge needle, compared to protein lysates
obtained with RIPA buffer plus sonication or using a commercial lysis buffer. 5 ug of
protei lysates were resolved in 4-12% SDS/PAGE and stained with SilverQuestTM silver
staining kit (Invitrogen, Karlsruhe, Germany) follwing the manufacturer’s instructions.
Stained gels were scanned on a GS 800 Densitometer®, and images processed with the
PDQuest® software, both from BioRad (Supplementary Fig S1a).
Western blot. Immunoblots were performed as described (5). Briefly, hepatocytes were
lysed in ice cold RIPA buffer (1x Tris-buffered saline, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS). Protein concentration was measured with DC protein assay
(Biorad, München, Germany). 30 of µg protein were resolved by SDS-PAGE using
NuPAGE Bis-Tris gels (Invitrogen, Karlsruhe, Germany) and transferred to nitrocellulose
membranes. Immunoblotting proceeded with incubations on primary antibodies over
night at 4°C, followed by secondary antibodies for 1h at room temperature as described.
Densitometric analysis of western blot bands was performed as described (6)
To validate western blot for signal transduction analysis on both CM and CS, we
compared protein lysates obtained as described in the SDS/PAGE silver staining section,
from hepatocytes in CS, untreated or stimulated for 1h with 5 ng/mL TGF-, to a protein
lysate obtained from CM cultured hepatocytes stimulated with the same dose of TGF-.
pSmad2 and total Smad2 levels were comparable in all conditions tested (Supplementary
Fig S1b).
Immunoprecipitation. Focal adhesion kinase (FAK) was immunoprecipitated from CM
or CS protein lysates in RIPA buffer as previously described (1). Briefly, 10 µg of rabbitanti FAK were added to 500 µg of protein lysates, plus 20 µL of 50% Protein-G beads
slurry (Santa Cruz, Heidelberg, Germany), rotated over night at 4°C, washed 3 times
with ice cold RIPA, boiled for 5 minutes, resolved on 10% SDS/PAGE and transferred to
nitrocellulose membranes, followed by immunobloting of phospho-tyrosin and FAK.
Specificity of IP was tested using an isotype control IgG (Supplementary Fig S2)
Adenovirus preparation, quantification and infection. Adenovirus vectors were
expanded, purified and quantified as described (5). Infections were done in CM and CS
hepatocytes 4h after plating, for 1h with 100 MOI of Adenovirus vectors expressing
constitutive active H-Ras-61L (7) (a kind gift from Thomas Gajewski, Dept Pathology and
Medicine, University of Chicago, USA), constitutive active HA-myr-Akt (8) (a kind gift
from Kenneth Walsh, Mol. Cardiology, Boston University School of Medicine, USA), GFP
and –galactosidase (9). The medium was then removed and the cells washed 3 times
with HBBS. For CS cultures, a second layer of collagen gel was added after infections,
followed by addition of serum free William’s E medium plus 100 nM Dexamethasone, on
both CM and CS cultures. We validated infectivity over the majority of the cells by using
an Adenovirus vector expressing GFP (Supplementary Fig S7). Infection with Adenovirus
vectors did not alter responsiveness to TGF-, as observed by induction of Smad2
phosphorylation and CTGF induction in AdLAcZ cells compared to non infected cells
(Supplementary Fig S8). Conversely, we validated transgene over expression and
functionality by using AdGFP, which led to strong GFP over expression (Supplementary
Fig S3).
DNA laddering. Genomic DNA fragmentation was analyzed as described (10). Briefly,
AML12 cells were collected in lysis buffer after indicated treatments and incubated with
proteinase K for 4 hrs at 37°C. Proteins were precipitated by adding 4 M NaCl at 4 °C for
1 hrs and centrifuged at 14.000 rpm for 60 min at 4°C. DNA was isolated with
phenol/cholophorm/isopropanol (Roth, Germany). 5 µg of DNA were resolved in 1.5 %
agarose gels and stained with ethidium bromide.
Nuclear condensation and fluorescent microscopy. Chromatin condensation was
analyzed as described (11). Briefly, hepatocytes on CS or CM were fixed in 4% PFA for
10 minutes, and nuclei were stained with 5 µg/mL Hoescht H33342 in PBS for 10
minutes. Phase contrast and fluorescent images were taken as described (2).
Hepatocyte viability and bile canaliculi function. Cell viability and bile canaliculi
function was analyzed by CMFDA export as described. Briefly, hepatocytes cultured on
CM or CS were incubated with 1 µM CellTrackerTM Green CMFDA (Invitrogen) for 30
minutes, then washed 3 times in serum free medium and photographed immediately,
using a . Phase contrast and fluorescent pictures were taken as described (2).
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