Supporting Materials and Methods
Cell lines, tissues and transfection experiments
HCC cell lines with stepwise metastatic potential (HCCLM3, MHCC97L, SMCC-7721, Bel-7402 and
Hep3B) were used in this study. HCCLM3 and MHCC97L generated on the same genetic background
were established at our institute. HCCLM3 and MHCC97L cells were maintained in Dulbecco’s modified
MEM medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin.
SMCC-7721, Bel7402 and Hep3B cells were maintained in MEM (minimum essential medium Eagle)
containing the same supplements as above.
Specimens taken from areas adjacent to the margin of tumors were collected from 403 consecutive
patients with HCC who underwent curative resection between 2000 and 2002 at the Liver Cancer
Institute of Fudan University (Shanghai, China). Paraffin blocks were selected only on the basis of the
availability of suitable formalin-fixed, paraffin-embedded tissue and complete clinicopathologic and
follow-up data for the patients. The histopathological diagnosis was based on the World Health
Organization criteria. Ethical approval was obtained from the Zhongshan Hospital Research Ethics
Committee, and written informed consent was obtained from each patient.
The pU6-vshRNA-CMV-RFP-1 lentiviral vectors (pU6 is a lentiviral vector) were purchased from
Shanghai GeneChem Co., and the vshRNA/siRNAs were constructed and synthesized by Shanghai
GeneChem Co., Ltd. The pAcGFP1-C1-Cryab cDNA plasmids were kindly provided by Professor Jack
Liang (Brigham and Women's Hospital, Boston) and Yong-Ting Wang (Med-X Research Institute,
Shanghai Jiao Tong University, Shanghai, China). The lentiviral vector and plasmid were transfected
into cells according to the manufacturer’s instructions. Transfection of the siRNAs was performed with
Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Cell Matrigel invasion assays, MTT assay and in vivo metastasis assays
Cell invasion assays were performed using 24-well transwells (8 μm pore size; Corning) precoated with
Matrigel (Falcon354480; BD Biosciences). A total of 1 × 10 5 cells were then suspended in 500 μl DMEM
containing 1% FBS and added to the upper chamber, and 750 μl DMEM containing 10% FBS was
placed in the lower chamber. After 48 h incubation, Matrigel and any remaining cells in the upper
chamber were removed by cotton swabs. Cells on the lower surface of the membrane were fixed in 4%
paraformaldehyde and stained with Giemsa. Cells in 5 microscopic fields (magnification, ×200) were
counted and photographed. All experiments were performed in triplicate.
Cells were aliquoted into a 96-well plate (2000 cells in 200 μl per well) and were incubated in medium
supplemented with sorafenib (0, 1, 5, 10 or 20 μM) provided by Bayer Pharma AG (Berlin, Germany) for
24 h. Twenty microliters of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) bromide
solution were added at the indicated time point, and the aliquots were incubated for 4 h. Medium was
replaced with 150 μl of dimethyl sulfoxide, and the aliquots were shaken for 10 min. Absorbance at 560
nm was measured to determine the number of viable cells in each well. All experiments were performed
in triplicate.
The in vivo metastasis assays were performed using nude mice. Athymic nude mice were obtained from
Shanghai Institute of Material Medicine and maintained in a pathogen-free environment. Animal care
and experimental protocols were performed in accordance with the guidelines established by the
Shanghai Medical Experimental Animal Care Commission. Ethical approval was obtained from the
Zhongshan Hospital Research Ethics Committee. HCC cells were injected intrahepatically with a 27-
gauge needle. At six weeks, six nude mice in each group were euthanized by anesthesia overdose and
visceral organs, including the lungs and livers. Tissues were histopathologically examined and
dehydrated/embedded in paraffin to generate serial sections. The tumor volume was calculated using
the following formula: V = π / 6 × (larger diameter) × (smaller diameter)2. Lung metastases were
examined and classified. H&E, Cryab, E-cadherin, Vimentin and Slug in vivo expression in excised
tumors were examined in serial tissue sections.
RNA expression analysis, Western blot Analysis, immunofluorescence assay and confocal
immunofluorescence.
Gene amplification and detection were performed using the ABI PRISM 7900 Sequence Detection
System (Applied Biosystems, Foster City, CA, USA) starting with 1µl cDNA and SYBR Green Realtime
PCR Master Mix (Takara, Japan). All transcript levels were normalized to Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) expression. Each sample was tested in triplicate. The relative expression was
analyzed by the comparative cycle threshold (Ct) method, according to the equation: 2 -ΔCt [ΔCt = Ct- Ct
(GAPDH)]. Reaction products of semiquantitative PCR analysiswere run out on a 2% agarose gel for
semiquantitative detection by autoradiography. All experiments were performed in triplicate.
Protein extracts from cells were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred onto polyvinylidene difluoride membranes, and incubated with the
corresponding antibodies. The membranes were developed using the enhanced chemiluminescence
method (Pierce, Rockford, IL, USA).
Cells were permeabilized with 0.1% Triton X-100 for 15 min at room temperature, washed with PBS,
and blocked with PBS containing 5% bovine serum albumin (BSA) for 1 h at room temperature. Cells
were treated with antibody overnight at 4 ℃. Cells were rinsed with PBS and then incubated with
secondary antibody for 1 h at room temperature. The slices were counterstained with diamidino
phenylindole (DAPI) and examined using fluorescence microscopy (Leica Microsystems Imaging
Solutions, Cambridge, UK).
Immunoprecipitation (IP) assays
Cells were washed twice with ice-cold PBS and then lysed with RIPA lysis buffer supplemented with
complete protease Inhibitor (Roche). After removing the insoluble material by centrifugation 12,000×g,
the precleared lysates were incubated with primary antibody pre-absorbed protein A- and G-Sepharose
beads overnight at 4°C. The precipitates were washed with the lysis buffer five times, boiled in 5xSDS
sample buffer for 5 min, and the proteins were resolved by SDS-PAGE on 10% gradient gels.
Subsequent immunoblots were probed with the appropriate antibody and detected by ECL.
Immunoisolation of Cryab-containing complexes, in-gel tryptic digestion and two-dimensional
liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS)
Immunoprecipitation was performed as described above. The solubilized extracts were then incubated
with 50μl of protein A/G beads coupled to a rabbit anti-human monoclonal Cryab antibody at 4℃
overnight. The beads were then washed with lysis buffer five times, followed by boiling in 5× buffer. The
immunoprecipitates were resolved on an SDS-PAGE denaturing gel, visualized by Coomassie blue
staining, and the protein band of interest was removed for MS analysis. MS was performed under 19-kV
accelerating voltage in reflection mode with an m/z range of 400 to 2,000. All MS/MS data were
identified using SEQUEST (v.28, Bioworks 3.3 software package, Thermo Electron) against the Human
International Protein Index (IPI) database (IPI human v3.45 FASTA with 71983 entries).