MATERIALS AND METHODS

Cell culture

HepG2 cell line was purchased from American Type Culture Collection (Manassas, VA)

in July 2009, and maintained as subconfluent monolayers in DMEM (Invitrogen) with 10% fetal

bovine serum (Hyclone, Logan, UT) and 100 units/ml penicillin plus 100 μg/ml streptomycin

(Invitrogen) at 37 °C with 10% CO2.

Transient Transfection and siRNA-mediated ROCK knock-down

ROCK siRNA (target sequence, 5�-CCATCAACGTGGAGAGCTT-3�) was synthesized

by Dharmacon Research, Inc. (Boulder, CO) based on previously studies (e.g., ref. #1). In

brief, HepG2 cells were transfected with the siRNA oligonucleotide or a control scramble

oligonucleotide using Lipofectamine 2000 per user manual (Invitrogen), and the efficiency of

siRNA-mediated protein suppression was assessed by Western blotting analysis. This siRNA

oligonucleotide effectively knocked down both ROCK1 and ROCK2 in several human cell lines

including HepG2 cells.

Twenty-four hours after the transfection, the invasion assay was then performed as

described below.

Tumor sample collection

Hepatocellular carcinoma and metastasis was diagnosed on the basis of typical clinical

and radiological findings, and also confirmed by pathology. The histological definition of

hepatocellular carcinoma was based on the classification proposed by the World Health

Organization. Tissues Samples were obtained with informed consent from 21 primary

HBV-HCC and metastatic patients (9 with intrahepatic metastasis, 9 with intraportal vein

thrombi [embolus] and 3 with lymph nodes metastasis) who underwent HCC radical resection

between 2006 and 2010 at Department of Hepatobiliary Surgery, Xijing Hospital. The

protocol was approved by the Ethics Committee of Xijing Hospital. Data does not contain any

information that may lead to the identification of the patients. Matched pairs of primary HCC

tissue samples, metastatic tumor and intraportai vein thromi samples from the same patient

were collected. Tissue specimens were divided into two parts: one was fixed promptly with 4%

formaldehyde solution, embedded in paraffin for histological study; the other was preserved in

liquid nitrogen immediately for proteomic study.

Immunohistochemistry

Deparaffinized, citrate buffer-treated normal tissue and liver sections were blocked with

3% hydrogen peroxide and incubated with mouse monoclonal antibody 6H11 against ezrin

(1:500 dilution) and a rabbit antibody against phospho-Thr567 (Cell Signaling Biotechnology;

1:200 dilution) followed by detection with the Envision system (Dako, Carpinteria, CA) and

hematoxylin counterstaining. Phospho-Thr567 levels were scored by staining intensity, and

the proportion of cells was stained without knowledge of patient outcome.

Immunofluorescence microscopy

For immunofluorescence, liver sections were then fixed in freshly prepared 4%

paraformaldehyde in PBS and rinsed three times in PBS. Cells on the coverslips were blocked

with 0.05% Tween 20 in PBS (TPBS) with 1% bovine serum albumin (Sigma). These cells

were incubated with various anti-ezrin antibodies in a humidified chamber for 1 h and then

washed three times in TPBS. Phospho-ezrinT567 was labeled with Alexa-488 conjugated

secondary antibody while ezrin protein was labeled with rhodamine conjugated

goat-anti-mouse antibody. Coverslips were supported on slides by grease pencil markings

and mounted in Vectashield (Vector Laboratories). Images were taken with a laser scanning

confocal microscope (LSM510; Zeiss) using a 40 x 1.4 numerical aperture PlanApo objective.

Figures were constructed using Adobe Photoshop.

Two-dimensional electrophoresis of hepatocellular carcinoma samples

Primary hepatocellular carcinoma tissue and metastatic embolus samples were

dissected and then labeled with Cy3 and Cy5, respectively, using Ettan DIGE Kit (Amersham

Biosciences) according to manufacturer’s manual. Following the labeling, the Cy3 and Cy5

labelled proteins were then mixed and precipitated with 85% alcohol and solubilized with lysis

buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% DTT, 2% IPG buffer [pH 4–7] (Amersham

Biosciences), 1 mM benzamidine, 2 μg/ml pepstatin-A, 20 μg/ml leupeptin, 10 μg/ml aprotinin,

1 mM sodium vanadate, 1 μM microcystin-LR). The protein samples were clarified by

centrifugation at 100,000g for 20 min before loading for IEF. ReadyStrips (pH 3~10) were

loaded with 100 μg whole-cell extract for analytical 2D gels and allowed to rehydrate for 18 hr

at room temperature. A gradient of 300–3500 V was applied to the strips followed by constant

3500 V, with focusing complete after 80,000 Vh. Prior to the second dimension, strips were

incubated (15 min) in equilibration buffer (6 M urea, 2% SDS, 0.375M Tris [pH 8.8], 30%

glycerol), first with 65 mM DTT (reductive) and second with 243 mM iodoacetamide (alkylating).

Equilibrated strips were then inserted onto gradient SDS–PAGE gels (8 x 10 cm; 6%–16%)

followed by electrophoresis. Two dimensional separated protein spots were qualified using

PhosphoImager (Typhoon, Amersham Biosciences).

Preparation of samples for mass spectrometry

Excised two-dimensional protein spots were destained, chopped into small fragments

with a razor blade, and subjected to digestion by modified porcine trypsin (50–100 ng/digestion;

Promega, Madison, WI) according to Fang et al. (2006). Peptides were recovered by three

extractions of the digestion mixture with 50% acetonitrile plus 5% trifluoroacetic acid and

desalted and concentrated using C18 ZipTips (Millipore Corp., Bedford, MA), eluting peptides in

50% (v/v) acetonitrile/water. All supernatants were pooled and concentrated to 5 μl in a

Speedvac and brought back up to 25 μl in 50% acetonitrile, 5% trifluoroacetic acid. The peptide

mix was stored at 20 °C until analysis.

Mass spectrometric identification of ezrin and its phosphopeptides

Aliquots of unseparated tryptic digests were co-crystallized with cyano-4-hydroxycinnamic

acid and analyzed using a MALDI delayed extraction reflectron TOF instrument (Bi-flex;

Bruker-Daltons, Framingham, MA) equipped with a nitrogen laser. Measurements were

performed in a positive ionization mode. All MALDI spectra were externally calibrated using a

standard peptide mixture (Sigma).

Data base interrogations based on experimentally determined peptide masses were

carried out using mass spectrometry (MS)-Fit, and PSD data interrogation was performed

using MS-Tag; both software programs were developed in the University of California San

Francisco MS Facility and are available on the World Wide Web at prospector.ucsf.edu. Both

the National Center for Biotechnology Information protein data base and Swiss-Prot data base

were searched. Search parameters included the putative protein molecular weight and a

peptide mass tolerance of 100–200 parts/million.

Confocal microscopy

To confirm if ezrin Thr567 is hyperphosphorylated in metastatic tumor, paired

hepatocellular carcinoma tissue sections were double stained with a mouse monoclonal

antibody 4A5 and phospho-Thr567 rabbit antibody. The doubly stained samples were

examined under a laser-scanning confocal microscope LSM510 NLO (Carl Zeiss) scan head

mounted transversely to an inverted microscope (Axiovert 200; Carl Zeiss) with a

40 × 1.3 numerical aperture PlanApo objective. Optical section series were collected with a

spacing of 0.4 μm in the z-axis through the ~12-μm thickness of the cell-in-cell complex. The

images from triple labeling were simultaneously collected using a dichroic filter set with Zeiss

image processing software (LSM 5, Carl Zeiss). Digital data was exported into Adobe

Photoshop for presentation.

Invasion assay

Matrigel-precoated Transwell chambers with PET membranes containing 8-μm pores

(BD Biosciences) were soaked in DMEM and incubated for 60 min at 37°C. After the chambers

were rehydrated, 1 x 105 cells in 0.5 mL serum-free culture medium were added to the upper

compartment of the Transwell chamber. DMEM (0.5 ml), supplemented with 10% FCS as a

chemoattractant, was added to the lower chamber. As a control, an equal number of uncoated

BD control chambers were seeded with cells in parallel. After 24 hours of incubation,

non-invaded cells in the upper compartment were removed using a cotton-tipped swab.

Invaded cells were stained with the Diff-Quik stain kit (BD Biosciences) and photographed (x20

magnification). Cells were counted in three unique fields of each triplicate membrane. Data

were expressed as the percentage of cells that invaded through the Matrigel matrix-coated

membrane relative to the cells that migrated through the control membrane.

For testing the role of phospho-ezrin mutants in cell migration, adenoviral infections of

HepG2 cells were performed 5 hours postplating. Cells were grown to 75% confluency and

infected with the control virus alone or the recombinant adenoviral constructs incorporating wild

type CFP-ezrin, phospho-mimicking CFP-ezrinT567A, and non-phosphorylatable CFP-ezrinT567A.

In general, the infection was executed by using 3 x 106 particles/ml of viruses. Cultures were

incubated at 37 °C for 12 h and then changed to fresh medium without viruses. The invasion

assays were then initiated. Data were derived from four independent experiments of triplicates.

To probe for the role of Rho kinase in regulating ezrin phosphorylation and cell invasion,

aliquots of HepG2 cells expressing CFP-ezrin (wild type) was treated with Y27632 (Sigma) at

appropriate concentrations (1-10 µM) for 2 hours before being seeded on the opposite side of

the Transwell from the matrigel for the invasion assay. To understand the mechanisms

underlying an increased invasive capability in HepG2 cells overexpressing wild type ezrin, we

employed siRNA to suppress endogenous ROCK protein level and examined protein level of

ROCK with correlated Thr567 phosphorylation of CFP-ezrin and invasive capability of treated

cells. The labeling of HepG2 cells with CFP-ezrin enables our assessment of invasion assay

with ease. However, those ezrin-overexpressing cells bear greater invasion capability

compared to that of parent cells.

We chose our experimental conditions based on the level of expression of CFP-ezrin

proteins as determined by intensity of fluorescence measured with a Spex fluorometer and the

general appearance of the cells. Direct observation of CFP and subsequent immunostaining

indicated that 92 ± 4% of HepG2 cells were expressing the ezrin constructs.

Tumor xenografts

Heptacarcinoma MHCC97-H cells (5x106 in 0.1 ml PBS) infecting with adenovirus

carrying CFP, CFP-ezrin, CFP-ezrinT567A, CFP-ezrinT567D were inoculated into the liver of

female NOD/SCID mice (6 weeks old; 10 animals per group) using an established protocol2.

Tumor growth was evaluated by monitoring tumor volume every 2 days for 10 weeks. The

MHCC97-H cell line was selected based on its reproducibility of metastasis in NOD/SCID

mice3. The animals were then sacrificed and the tumor xenografts, livers of the mice were

harvested for further evaluation. Cryosections (4 �m) of the harvested organs were stained

with hematoxylin and eosin (HE) for histological assessment and immunocytochemistry of

human CFP-ezrin expression. The protocol was approved by the Animal Using Committee of

Xijing Hospital.

Early autopsy revealed that human hepatocellular carcinoma often spread within liver4.

Since ROCK is implicated in intragepatic metastasis of HCC, we attempted to probe for

possible metastasis of liver cancer to distinct organs such as lung. To this end, the lungs of

tumor-bearing mice infected with CFP, wild type ezrin (WT), ezrin-T567A (T567A) and

ezrin-T567D (T567D) were examined and cryosections (4 �m) of the harvested organs were

stained with hematoxylin and eosin (HE) for gross histological assessment.

SDS-PAGE and Western blotting analyses

Samples were subjected to SDS-PAGE and transferred onto nitrocellulose membrane.

Proteins were probed by appropriate primary antibodies and detected using ECL (Pierce).

To probe for the nature of ezrin phosphorylation, samples dissected from normal and

hepatocarcinoma tissues were harvested for SDS-PAGE gel fractionation followed by

transferring onto a nitrocellulose membrane. The phosphorylation of ezrin was probed with an

anti-phospho-Thr567 antibody (Cell Signaling Technology, MA, USA). Blots were developed

using ECL, and the spot intensities were quantified using a Typhoon PhosphorImager

(Amersham Biosciences).

Statistical analysis

The descriptive statistics are provided with mean ±SE. t-test was used to assess the

experimental effects and differences. P<0.05 was considered statistically significant.

References: 1. Chevrier V, Piel M, Collomb N, Saoudi Y, Frank R, Paintrand M, Narumiya S, Bornens M, Job

D. The Rho-associated protein kinase p160ROCK is required for centrosome positioning. J Cell Biol. 2002. 157: 807-17.

2. Genda T, Sakamoto M, Ichida T, et al. Cell motility mediated by Rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma. Hepatology. 1999; 30:1027-1036.

3. Ding S-J, Li Y, Tang Y-X, et al. From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis. Mol Cell Proteomics 2004;3:73-81.

4. Yuki K, Hirohashi S, Sakamoto M et al. Growth and spread of hepatocellular carcinoma. A review of 240 consecutive autopsy cases. Cancer 1990; 66:2174-2179.

Supplemental FigureFigure S1

A. B.

Normal tissue HCC cancer embolus

Examples of primary HCC and metastatic samples taken from patients for proteomic analyses.Primary HCC and cancer embolus were dissected and dissolved in 2D SDS-PAGE sample bufferas described in the Methodsas described in the Methods.

Figure S2

MHCC97-H cells were infected with adenovirus containing CFP-WT, T567A or T567D Ezrinrespectively. After 16hr of infection, cells were collected and lysed in sample buffer. Imunoblottingwas performed with anti CFP antibody to identify the expression of each proteinwas performed with anti-CFP antibody to identify the expression of each protein.

Figure S3

MHCC97-H cells were infected with adenovirus containing T567D ezrinand injected into the livers of female NOD/SCID mice as described inthe Methods The animals werethe Methods. The animals weresacrificed and the livers of xenograftmice were fixed for immunocytochemistry of human CFP-ezrin expression.

Figure S4A B

CC

TA

TD

WTWT

A, representative images of the lungs of tumor-bearing mice infected with CFP, wild type ezrin (WT), ezrin-T567A (T567A) d i T567D (T567D) ti land ezrin-T567D (T567D), respectively.

B, H&E staining for the xenografts demonstrating that there are no metastasis in lung in the tumor-bearing mice infected with CFP, wild type ezrin (WT), ezrin-T567A (T567A) and ezrin-T567D (T567D), respectively. Both 100X images and magnified images (200X) were also present.