Supplementary methods
Reagents
The avidin-biotin-peroxidase complex (ABC) kit was purchased from Funakoshi Chemical Co.
(Tokyo, Japan). All other chemicals were from Sigma Chemical Co. (St. Louis, MO) or Wako Pure
Chemical Co. (Tokyo, Japan). The following antibodies were used: Antibodies to Shc and p-Shc
used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell
Signaling Technology (CST, Danvers, MA). Antibodies to Erk1/2, p-Erk1/2, SAPK/JNK,
p-SAPK/JNK, p38MAPK, p-p38MAPK, Snail, HSP90, HSP70, HSP27 and ß-actin were obtained
from CST (Danvers, MA), anti-human cytokeratin (CK) 7 and CK19 were from Abcam (Cambridge,
MA) and DakoCytomation (Carpinteria, CA) (Clone BA17) for staining, respectively. Anti-human
Ki67 antibody for immunohistochemistry was purchased from ZYMED laboratories Inc. (South
San Francisco, CA). Anti-human PCNA antibody was from Santa Cruz. Antibodies are listed in
Suppl. Table 1.
Quantitative PCR
Relative Transcript levels were quantified in total liver RNA by real-time RT-PCR on a LightCycler
1.5 instrument (Roche, Mannheim, Germany) using the TaqMan and SYBR Green methodologies
(LightCycler FastStart DNA Master SYBR Green I kit, Roche, Penzberg, Germany). TaqMan
probes (dual-labeled with 5’-FAM and 3’-TAMRA) and primers for major profibrogenic mRNA
quantitation were designed and extensively validated for target specificity and linearity of signal
amplification. Melting curve analysis and visualization of PCR-products on agarose gels were
performed to ensure specificity of RT-PCR. The housekeeping gene beta-2 microglobulin (β2MG)
was amplified in parallel reactions for normalization. Sequences of primers and probes used in this
study are summarized in Suppl. Table 2.
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Immunohistology and determination of Shc labeling indices
Formalin-fixed liver specimens were stained with primary Shc antibody, followed by the ABC
staining kit (Funakoshi Chemical, Tokyo, Japan) as described previously1. For signal amplification,
the Renaissance TSA amplification kit (NEN Life Science Products, Boston, MA) was used.
Sections were counterstained with Mayer's hematoxylin. Cytoplasmic labeling indices (LI, %) for
Shc-positive cells were determined by evaluating at least 1,000 hepatocytes or cancer cells in 10
random microscopic fields at x 400 magnification.
Cell and Tissue lysates, Western blotting and inhibition of Erk1/2
Cell cultures and snap frozen tissues were homogenized in lysis buffer [50 mM N-2
hydroxyethylpiperazine-N'-2- ethanesulfonic acid (HEPES; pH 7.0), 250 mM NaCl, 0.1% Nonidet
P-40, 100 mM NaF, 200 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 10
µg/ml aprotinin] and the lysates centrifuged at 29,000 g for 20 min at 4˚C. The protein
concentration of each sample was measured by the Bradford assay2. Due to limitation of biopsy
material, lysates from 3 different patients were pooled (groups of non-cirrhotic, of cirrhotic hepatitis
C, and HCC patients).
Western blot was performed according to standard procedures3, using optimal dilutions of primary
antibodies and horseradish peroxidase-labeled secondary antibodies. Immunoreactive proteins were
visualized with an enhanced chemiluminescence detection system (Amersham) and a radiographic
exposure time of 30 sec.
Cells cultivated in 10% FBS DMEM were exposed to 10 µM of the Erk1/2 inhibitor U0126 (CST,
#9903) starting at day 3 after heat treatment. Inhibitor and medium were renewed after 24 h. After
48 h RNA and protein were isolated. Densitometry was performed using ImageJ software (created
by National Institute of Health).
In situ quantification of in vivo HCC cell proliferation and apoptosis
2
Formalin-fixed and paraffin-embedded sections of tumor tissues were stained for proliferation (Ki67)
or apoptosis (in situ Cell Death Detection Kit, exploiting terminal deoxynucleotidyl transferase dUTP
nick end labeling [TUNEL], Roche, In). Positive cells/ high power field (HPF, x400 magnification)
were randomly counted in 5 different areas in the inner tumor mass, the invasion front and the
intermediate zone. Average numbers were calculated for each tumor and used for statistical
assessment.
Tumor invasion assay
The invasiveness of hepatoma cells was assessed based on the number of cells invading through
Matrigel-coated transwell chambers (BD Biosciences, Franklin Lakes, NJ) as previously described4.
Transwell inserts with 8 µm pores were coated with Matrigel (5 µg/well; BD Biosciences, Bedford,
MA). At day 3 after heating, hepatoma cells (4×104 cells/ml) were seeded in the upper chambers and
incubated for 48 hr. Cancer cells that invaded to the lower surface of the Matrigel-coated membrane
were fixed with 70% ethanol, stained with H&E, and counted in five random fields at 200×
magnification under a light microscope (AxioImager M1; Zeiss, Jena). For inhibition of Erk1/2, cells
were exposed to 10 µM of U0126 (CST) during the invasion assay. The results were expressed as the
mean number of invading cells. Each experiment was carried out in triplicate wells, and independent
experiments were repeated more than two times.
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References
1. Yoshida S, Masaki T, Feng H, Yuji J, Miyauchi Y, Funaki T, Yoshiji H, et al. Enhanced expression
of adaptor molecule p46 Shc in nuclei of hepatocellular carcinoma cells: study of LEC rats. Int J
Oncol 2004;25:1089-1096.
2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.
3. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A
1979;76:4350-4354.
4. Moriyama T, Ohuchida K, Mizumoto K, Cui L, Ikenaga N, Sato N, Tanaka M. Enhanced cell
migration and invasion of CD133+ pancreatic cancer cells cocultured with pancreatic stromal cells.
Cancer 2010;116:3357-3368.
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