Supplemental Methods
Array hybridization. Total RNA was isolated using RNeasy Mini Kit (Qiagen)
according to manufacturer’s conditions. Three RNA samples from SAMe-D and
mouse hepatocytes were hybridized to Mouse Expression MOE430 array
(Affymetrix) containing 22,690 entries, and the data analyzed by GeneChip
Operating Software (GCOS 1.4, Affymetrix). To define the set of SAMe-D
regulated genes, we compared experiments using a multivariate permutation t
test. In total, 2,746 unique features showed significant (p ≤ 0.001) and at least 2fold expression difference between the two types of samples.
FISH analysis. SAMe-D cells were exposed to Colcemid (0.02 µg/ml) for 4h,
trypsinized and treated with 0.06 M KCl hypotonic solution for 30 minutes. Then,
the cells were fixed in methanol: acetic acid (3:1), and dropped onto wet slides.
Metaphase spreads from SAMe-D cells were simultaneously hybridized with a
chromosome 11 painting probe labelled with Biotin-dUTP and a probe prepared
from the BAC clone CH29-485M16 (C.H.O.R.I.) labelled with Digoxigenin-dUTP,
which include the whole sequence of TP53. Cell preparation was hybridized
following standard procedures (1). The digoxigenin and the biotin labelled probes
were detected with FITC-conjugated sheep anti-digoxigenin antibodies, and
streptavidin conjugated with Texas Red, respectively. Specimens were examined
with an Olympus microscope equipped with epifluorescence optics and a cooled
CCD camera. Images were generated and analyzed using the software provided
by the manufacturer.
P53 sequencing. Genomic DNA from SAMe-D cells was isolated by using
NucleoSPIN Kit (Macherey-Nagel) and amplified using Platinum Taq DNA
Polymerase High Fidelity (Invitrogen). Primer sequences are detailed in
Supplementary table V. PCR products were run on 1% agarose gel, extracted
and purified using QIAquick Gel Extraction Kit (Qiagen) according to instructions
provided and DNA was sequenced.
Fas-mediated apoptotic cell death. SAMe-D cell line and primary mouse
hepatocytes were cultured with or without 2 µg/mL of Fas agonist Jo2 antibody
(BDbiosciences) in 0.1% FBS-medium and total protein extracts were analyzed
by Western blot.
Supplemental Figure legends
Supplementary Figure 1. Proteins regulated in SAMe-D cell line. Cell extracts
were collected and analyzed via Western blotting with the indicated antibodies.
SAMe-D cell line shows differential expression of (A) p53 and apoptosis signaling
pathways, and (B) kinases and cell cycle proteins, compared to hepatocytes.
Data are representative of an experiment performed four times.
Supplementary Figure 2. Analysis of the second clone (Clone 2) isolated from
MAT1A-KO mice HCC. Total (A) and cytosolic (B) cell extracts were collected
and analyzed via Western blotting with the indicated antibodies. (A) The Clone 2
shows similar protein expression pattern than the SAMe-D cell line compared to
WT hepatocytes. In both tumor clones, (B) Western blot analysis of cytosolic
extracts revealed p-LKB1 Ser(428) mainly located in the cytosol as well as (C) an
overexpression of cytosolic p53 protein analyzed by immunocytochemistry.
Supplementary Figure 3. TP53 copy number analysis by fluorescence in situ
hybridization (FISH). FISH analysis of TP53 gene copies (green) in chromosome
11 (red) in SAMe-D cell line nuclei. DNA counter staining with DAPI is in blue. (A)
Metaphase cell showing four copies of chromosome 11 harboring one TP53
signal each. (B) Four interphase nuclei showing the 4 copies of the TP53 gene.
Supplementary Figure 4. Specificity of immunolabelling of p53 and pp53 (Ser
389) antibodies used. (A) SAMe-D cells were immunolabelled with DAPI and p53
antibody. (B) SAMe-D cells transfected with control and p53 siRNA, were
immunolabelled with p53 and p-p53 (Ser389) antibodies. (C) SAMe-D cells were
treated with leptomycin (20 nM) during 1 hour and p53 and HuR proteins were
detected by immunocitochemistry. Images of the cells were taken with a 60x
objective using a Leica TCS-SP (UV) confocal laser microscope. Data are
representative of an experiment performed four times.
Supplementary Figure 5. Graphical representation (mean standard error of the
mean [SEM]) of the densitometry changes of the experiment represented in
Figure 2B. *P < 0.05 Control versus UVC treated cells, and **P < 0.05 Ly or Wo
versus UVC treated cells (n=3).
Supplementary Figure 6. Graphical representation (mean standard error of the
mean [SEM]) of the densitometry changes of the experiment represented in
Figure 2C. *P < 0.05 Indicated silencing and treated cells versus silenced control
untreated cells (n=3).
Supplementary Figure 7. Role of AMPK in AKT phosphorylation in SAMe-D cell
line after UVC light (A) Mouse hepatocytes and SAMe-D cells were incubated for
1 hour in the presence or absence of LY204002 (10 µM) or Wortmanin (100 nM),
before the treatment with UVC light (20J) during 1 hour. Cytosolic cell extracts
were collected and analyzed via western blotting with the pAMPK1, AMPK1
and GAPDH antibodies. (B) SAMe-D cells transfected with control or AMPK
siRNA, were incubated for 1 hour in the presence or absence of Wortmanin (100
nM), before the treatment with UVC light (20J) during 1 hour. At the end of this
period cytosolic protein extracts were analyzed via western blotting with the
indicated antibodies.
Supplementary Figure 8. Sensitivity to Fas-mediated apoptosis in primary
hepatocytes compared to SAMe-D cell line. Primary mouse hepatocytes and
SAMe-D cells (A) were treated with Jo2 antibody (2 µg/mL) and cells were
lysated at the indicated times. (B) SAMe-D cells were supplemented every 24
hours with Jo2 antibody (2µg/mL) and lysated at the indicated times. Whole cell
lysates were analyzed by Western blot and incubated with the indicated
antibodies.
Supplementary Figure 9. HAUSP localization after UVC treatment in mouse
hepatocytes compared to SAMe-D cell line. Mouse hepatocytes and SAMe-D
cells were treated with UVC light (20J) at the indicated times and HAUSP protein
was detected by immunostaining. Images of the cells were taken with a 60x
objective using a Leica TCS-SP (UV) confocal laser microscope. Data are
representative of an experiment performed four times.
Supplementary Table I: Conditions of the antibodies used for Western blot
analysis. After blocking, membranes were incubated overnight at 4ºC with
specific antibody. Optimal incubation conditions, appropriate concentration, and
supplier for each specific primary antibody are listed above. This was followed by
1 hour of incubation with secondary antibodies conjugated to horseradish
peroxides. Immunoreactive proteins were detected by Western lightning
Chemiluminescence Reagent (PerkinElmer, Boston).
Supplementary
Table
II:
Conditions
of
the
antibodies
used
for
immunocytochemical analysis. Table described the appropriate concentration,
supplier and fixation conditions for each specific antibody used in the
immunocytochemical study.
Supplementary Table III: siRNA sequences used for target gene silencing in
SAMe-D cell line.
Supplementary Table IV: Genes regulated in SAMe-D cell line. Fold changes
between the hybridization intensity of the SAMe-D cell line compared to primary
mouse hepatocytes samples.
Supplementary Table V: Oligonucleotide primers used for P53 sequencing
analysis performed in SAMe-D cell line.
Supplemental references
1. Parada LA, McQueen PG, Misteli T. (2004). Tissue-specific spatial
organization of mammalian genomes. Genome Biology 5, R44.