SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Ectopic HNF4α expression in Hep3B hepatoma cells induces an
epithelial phenotype, inhibits invasive behaviour and down-regulates mesenchymal markers
(A) Phase-contrast micrographs showing the morphological changes observed in BW-H4 cells.
Scale bar = 40 m.
(B) Representative phase-contrast micrographs of a wound healing assay comparing BW-H4 to
mock-infected BW1J at the indicated times. Scale bar = 80 m
(C) Histogram of invasivity assay. Data are represented as mean and SD of triplicated
experiments (P<0.01 Student t test).
(D) Wound healing assay of Hep3B hepatoma cells showing after 24 hours a reduced migration
of cells expressing HNF4α (3B-H4) compared to mock-infected cells. Scale bar = 80 m
(E) qPCR
analysis
for
Phosphoenolpyruvate
the
indicated
carboxykinase,
genes
(HNF1,
AldoB=AldolaseB,
APOC3=apolipoproteinC3,
PEPCK=
Cdh1=E-cadherin,
Krt18=Cytokeratin 18, JUP= Junction Plakoglobin, Cldn1=Claudin1, Vim=Vimentin,
Fn1=Fibronectin, Mmp2=Metalloproteinase 2).
Supplementary Figure 2. HNF4α induces direct transcriptional repression of human Snail
gene.
(A) qPCR analysis of endogenous Snail mRNA level. The values are calculated by Ct
method and expressed as fold of change in gene expression in 3B-H4 versus parental
Hep3B cells ( *P <0.05 Student t test) .
(B) Western blot analysis of Snail, HNF4α and, as control, Tubulin in parental Hep3B and 3BH4 cells .
(C) EMSA assays on HNF4α consensus binding sites of human apoC3 and Snail promoters
were performed with the indicated nuclear extracts and probes.
(D) qPCR-ChIP assay with anti HNF4α and anti-NCoR antibodies on chromatin from 3B-H4
cells. As controls, ChIPs were also performed with unrelated IgG. Consensus sites for
1
HNF4α, schematically depicted as black boxes (H4-2 and H4-1) and the unrelated
sequences used as negative control (ns), were amplified with primers depicted as arrows and
listed in supplementary TABLE 5. Mean and SD values from three independent experiments
are reported with statistical significance (*P<0.05).
(E) EMSA assays on HNF1α consensus binding site (-879/-862) of human Snail promoter were
performed with the indicated nuclear extracts. Probes are reported in Supplementary
TABLE 1.
Supplementary Figure 3. Positions of HNF4α and HNF1α putative binding sites on
mesenchymal genes promoters
(A) Consensus sites for HNF4α on Mus musculus fibronectin promoter analyzed in qPCR ChIP
assays are schematically depicted as black boxes (H4) and black arrows indicate the
amplified region and the unrelated sequences used as negative control (ns).
(B) Consensus sites for HNF4α and HNF1α on Mus musculus vimentin promoter and the
unrelated sequences used as negative control (ns) in qPCR ChIP assays are schematically
depicted as in 3A.
(C) Consensus sites for HNF4α and HNF1α on Mus musculus desmin promoter and the
unrelated sequences used as negative control (ns) in qPCR ChIP assays are schematically
depicted as in 3A.
Supplementary Figure 4. HNF4α knockout mice express mesenchymal genes
qPCR analysis of the indicated genes in wild-type floxed Alb- Hnf4α (Alb- Hnf4α F/F) and AlbHnf4α KO (-/-) mice calculated by Ct method (* P<0.05, ** P<0.01 Student t test). Mean data of
three different animals per group.
Supplementary TABLES Legend.
Supplementary TABLE 4
2
For each qPCR ChIP primers to the corresponding coding sequences were used as negative control
genomic regions (ns). The primers are the same listed in Supplementary TABLE 3. Primers
sequences for HNF4 target FXIIIB were previously reported 1.
3
4
5
6
7
8
9
10
11
12
SUPPLEMENTARY METHODS
Cell culture conditions
BW1J and Hep3B cells were grown in DMEM with 10% FBS (Cambrex BioSciences, Verviers,
Belgium), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO) and antibiotics. MMH-D3
hepatocytes were grown in RPMI 1640 with 10% FBS (GIBCO), 50 ng/ml EGF, 30 ng/ml IGF II
(PeproTech Inc, Rocky Hill, NJ), 10 g/ml insulin (Roche, Mannheim, Germany) and antibiotics,
on collagen I (Upstate Biotechnology, Waltham, USA) coated dishes. When indicated cells were
treated with 4 ng/ml TGF (PeproTech Inc., Rocky Hill, NJ, USA) for 48 hours.
Plasmids and production of recombinant retroviruses
For forced expression experiments, the rat HNF4α and HNF1α cDNAs were subcloned into the
pBABEpuro retroviral vector. To produce recombinant replication incompetent retroviruses, 293GP
packaging cells were transiently transfected according to standard procedures with the HNF4α and
HNF1α retroviral constructs together with VSV envelope protein encoding plasmid. Viral particles
were collected 48 and 72 hours after transfection. Stable HNF4α over-expressing cells and control
cells were obtained by infection with pBABE Flag-HNF4α orempty vector viral supernatant,
respectively, and selected in 3 g/ml puromycin for a week.
Site-directed mutagenesis.
Mutations in each of the putative HNF4α and HNF1α binding sites on the murine Snai1 promoter
were generated by site-directed mutagenesis using the QuickChange XL Site-Directed Mutagenesis
kit (Stratagene, La Jolla, CA) as indicated by the manufacturer. The point mutations in the
corresponding plasmids were verified by sequencing. Primers used to generate the desired mutants
are listed in Supplementary TABLE 1.
Luciferase assays
BW1J cells were transfected by Lipofectamine 2000 (Invitrogen, San Diego, CA) with Snail1
promoter (-900 to +21) fused to the firefly reporter gene 15 and pCLBCX-Flag-HNF4αor pCLBCX-
13
HNF1αexpression vectors. The cotransfected plasmid pCMV--galactosidase was used as control.
All transfections were performed in triplicate. Luciferase activity was measured 48 hours after
transfection with the Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI)
and normalized for both -Galactosidase activity and protein amount.
Migration and invasivity assays
For the wound healing assays, a scratch was made on a confluent cell monolayer with a 200-μl
pipette tip. For transwell invasivity assays, 8 m pore 24-well cell culture plates (Corning Inc, NY,
USA) coated with type I collagen (0.1 mg/ml; Upstate Biotechnology, CA, USA) were used. Equal
numbers of cells (25,000/well) were plated onto the top surface of the transwell filter in the upper
chamber. The lower chamber contained 20 ng/ml IL-8 (Pierce, Chemical, Rockford, IL). Cells were
fixed after 24 hours with 4% paraformaldehyde, stained with Giemsa solution and DAPI, and the
nuclei counted manually in five random microscopic fields. Experiments were performed two times
in triplicate. For the wound healing assay in HNF4α knock-down MMH-D3, cells were plated in 35
mm plates and a scratch was made 24 hours after HNF4α siRNA or siRNA control transfection. A
low serum (2% FBS) culture medium was also added to the plates to inhibit cell proliferation. The
micrographs were taken at 24 hours or 48 hours after the scratch on paraformaldehyde-fixed cells
and the migrating cells were counted on 6 fields. Cells were also stained with DAPI to easily count
the nuclei of migrating cells crossing the scratch. The assay was repeated two times.
RNA extraction, Reverse Transcription and RT-qPCR
Total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA) or NucleoSpin® RNA II kit
(Macherey-Nagel, Germany) and reverse transcribed with MMLV-reverse-transcriptase (Promega,
MI, Italy). RT-qPCR was performed using BioRad Miniopticon with KAPA SYBR® Green FAST
qPCR mix (KAPABIOSYSTEMS, Woburn, MA, USA). Relative amounts were obtained with 2–
ΔΔCt
method and normalized to β-actin.
SDS-PAGE and Western Blots
14
Cells were lysed in RIPA buffer containing protease inhibitors (Roche, Monza, IT). Protein
concentrations were determined by use of the Bio-Rad assay reagent (Bio-Rad Laboratories,
Hercules, CA). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF
membranes (Millipore, MA, USA). Blots were blocked in 5% non-fat milk prepared in TBST and
incubated overnight with the primary antibody. Antibodies against HNF4α (C19, sc6556), αTubulin
(TU-02, sc8035), Snail (H-130, sc28199), Vimentin (sc-5565) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), α-SMA antibody (from Sigma-Aldrich, USA). Blots were then
incubated with HRP-conjugated species-specific secondary antibodies (Bio-Rad,Hercules, CA.
USA; Jackson Immunoresearch Laboratories, Inc. Baltimore, USA), followed by enhanced
chemiluminescence reaction (Pierce Chemical, Rockford, IL).
Electrophoretic mobility shift assays
For competition experiments, nuclear extracts were preincubated with a 200-fold molar excess of
wild-type or mutated cold probes. For supershift experiments, nuclear extracts were preincubated
with 2µg anti-HNF4α (sc8987), anti-HNF1α (sc8986X), or anti-Tubulin (sc8035), Santa Cruz
Biotechnology, as a non-specific antibody control.
ChIP analysis
Immunoprecipitations were performed with anti-HNF4α
-NCoR (sc 8994), anti-
HNF1α (sc 6547), the negative control rabbit IgG (sc-2027) Santa Cruz Biotechnology. A 1:10
dilution of starting chromatin DNA was used as template for input amplification. QPCR analysis
was performed in triplicate and repeated two independent times. All signals were normalized to
total input chromatin adjusted for diluition factor. Data are represented as fold enrichment of
immunoprecipitates on specific target genes subtracted of background signal (immunoprecipitates
with IgG) and compared to the amplification of non specific genomic regions (ns), used as negative
control.
For in-vivo ChIP analysis, livers were perfused with 2% w/v collagenase, dissociated in DMEM,
and filtered through 70 μm strainers prior to fixation in 1% formaldehyde. Fixation was stopped
15
with the addition of excess glycine. Chromatin preparations were made using the Cell Signaling
Simple ChIP Enzymatic Chromatin IP kit with magnetic beads. HNF4α was immunoprecipitated
using 3 μg of goat anti-HNF4α C19 (sc-6556, Santa Cruz Biotechnology, Inc.) overnight at 4ºC. 2%
input was reserved prior to immunoprecipitation. QPCR reactions were prepared using 1 μl DNA
and 200 nM primers with the Fast Sybr Green mix (Applied Biosystems) and run on a 7900HT Fast
Real-Time PCR machine (Applied Biosystems). qPCR ChIP primers are listed in Supplementary
TABLE 4 and 5.
Immunofluorescence and Immunohistochemistry.
Immunofluorescence analyses were performed on deparaffinized liver sections of Hnf4 F/F and
Hnf4 -/- mice. Briefly, formalin-fixed, paraffin-embedded mice liver samples were cut at 5 μM
thickness and mounted on polylysine-charged glass slides. The sections were deparaffinized and
rehydrated
in
a
series
of
graded
alcohol:water
solutions
and
then
incubated
for
immunohistochemistry with the primary antibodies anti-Desmin (ThermoFisher Scientific,Fremont,
USA), anti α-SMA (Sigma-Aldrich, St. Louis, USA), anti-Vimentin (Millipore, Billerica, MA).
Secondary antibodies were used in an indirect immunoperoxidase protocol as described previously2.
For confocal microscopy antibodies against αSMA (1:200) and Albumin (Novus Biologicals, Inc.,
Littleton,CO; 1:100) were used. AlexaFluor-conjugated secondary antibodies were used for signal
detection. Immunofluorescence analyses in MMH-D3 cells were performed with anti-Vimentin
(1:50), anti-Ecadherin antibody (BD Biosciences Pharmingen, Bedford, MA; 1:50), anti ZO-1
(Zymed Laboratories, San Francisco, CA; 1:50).
Supplementary References
1. Inoue Y, Peters LL, Yim SH, Inoue J, Gonzalez FJ. Role of hepatocyte nuclear factor 4alpha
in control of blood coagulation factor gene expression. J Mol Med. 2006 84(4):334-44.
2. Hsu, SM, Raine, L, Fanger, H. Use of avidin-biotin-peroxidase complex (ABC) in
immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP)
procedures. J. Histochem. Cytochem. 1981. 29:577-580.
16
17