Supplementary materials and methods Quantitative reverse

Supplementary materials and methods
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 µg
of total RNA was converted to cDNA with a First Strand cDNA Synthesis Kit (Amersham
Biosciences Corp., Piscataway, NJ, USA). PCR was performed with a SYBR Green PCR
Core Reagents Kit (Applied Biosystems, Foster City, CA, USA). Real-time detection of the
emission intensity of SYBR green bound to double-stranded DNA was performed with an
ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as described previously
[1]. ACTB-specific PCR products, which were amplified from the same RNA samples, served
as an internal control. Primer sequences and additional PCR conditions are available upon
Western blot
For Western blot analysis, tissue samples or cells were lysed as described previously [2]. The
lysates (40 µg) were solubilized in Laemmli sample buffer by boiling and then subjected to
10% SDS-polyacrylamide gel electrophoresis followed by electrotransfer onto a nitrocellulose
filter. The filter was incubated with the primary antibody against tetraspanin 8 (Abgent).
Peroxidase-conjugated anti-mouse IgG was used in the secondary reaction.
Immunocomplexes were visualized with an ECL Western Blot Detection System (Amersham
Biosciences). -actin antibody (Sigma Chemical, St. Louis, MO, USA) was also used as a
loading control.
Immunohistochemical analysis was performed with a Dako Envison+ Mouse Peroxidase
Detection System (Dako Cytomation, Carpinteria, CA, USA). Antigen retrieval was
performed by heating in Target Retrieval Solution, pH9 (Dako Cytomation) for 40 min at
97˚C. After peroxidase activity was blocked with 3% H2O2-methanol for 10 min, sections
were incubated with normal goat serum (Dako Cytomation) for 20 min to block nonspecific
antibody binding sites. Sections were incubated with mouse monoclonal anti-tetraspanin 8,
1:50 (Abgent, San Diego, CA, USA) or mouse monoclonal anti-CD31, 1:50 (a marker for
endothelial cell, Dako Cytomation) for 1 h at room temperature, followed by incubations with
Envision+ anti-mouse peroxidase for 1 h. For color reaction, sections were incubated with
DAB Substrate-Chromogen Solution (Dako Cytomation) for 10 min. Sections were
counterstained with 0.1% hematoxylin. A result was considered positive if at least 10% of the
GC cells were stained. When fewer than 10% of cancer cells were stained, the
immunostaining was considered negative.
Microvessel counts
Microvessel density was assessed by light microscopy in immunohistochemistry-stained areas
of tumor containing the highest numbers of capillaries and small venules at the invasive edge.
Highly vascularized areas were first identified by scanning tumor sections at low power (40x).
The vessel count was determined for three such areas at 400x and the mean of the three
counts was calculated. A vessel lumen was not necessary for a structure to be defined as a
RNAi and cell growth, in vitro invasion and wound healing assays
To knockdown endogenous TSPAN8, RNAi was performed. Short interfering RNA (siRNA)
oligonucleotides for TSPAN8 and a negative control were purchased from Invitrogen
(Carlsbad, CA, USA). Two independent oligonucletides were used for TSPAN8 siRNA.
Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) as described
previously [3]. Briefly, 60 pmol of siRNA and 10 µL of Lipofectamine RNAiMAX were
mixed in 1 mL of RMPI medium (10 nmol/L final siRNA concentration). After 20 minutes of
incubation, the mixture was added to the cells and then plated on dishes for each assay.
Forty-eight hours after transfection, cells were analyzed for all experiments. The cells were
seeded at a density of 2,000 cells per well in 96-well plates. Cell growth was monitored after
1, 2, 4 and 8 days by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay [4]. Three independent experiments were carried out. Mean ± SE was calculated for
each of the experiments. Modified Boyden chamber assays were performed to examine cell
invasiveness. Cells were plated at 10,000 cells per well in RPMI 1640 medium plus 1% serum
in the upper chamber of a Transwell Insert (8 µm pore diameter; Chemicon, Temecula, CA,
USA) coated with Matrigel. Medium containing 10% serum was added in the bottom
chamber. After two days, cells in the upper chamber were removed by scraping, and the cells
remaining on the lower surface of the insert were stained with CyQuant GR dye to assess the
number of cells. Three independent experiments were carried out. Mean ± SE was calculated
for each of the experiments. For the wound healing assay, cells were plated onto fibronectin
coated coverslips. The monolayer cells were then scratched manually with a plastic pipette tip,
and after being washed with PBS, the wounded monolayers of the cells were allowed to heal
for 24 h in RPMI 1640 containing 10% FBS.
Statistical methods
Correlations between clinicopathologic parameters and tetraspanin 8 protein expression were
analyzed by Fisher’s exact test. Kaplan-Meier survival curves were constructed for
tetraspanin 8-positive and tetraspanin 8-negative patients. Survival rates were compared
between tetraspanin 8-positive and tetraspanin 8-negative groups. Differences between
survival curves were tested for statistical significance by log-rank test. Univariate and
multivariate Cox regression was used to evaluate the associations between clinical covariates
and cancer-specific mortality. SPSS was used to perform these analyses (SPSS Inc., Chicago,
IL, USA). Hazard ratio (HR) and 95% confidence interval (CI) were estimated from Cox
proportional hazard models. For all analyses, age was treated as a categorical variable (≥ 65
years versus < 65 years). For final multivariable Cox regression models, all variables that
were moderately associated (P < 0.10) with cancer-specific mortality were included. A P
value of < 0.05 was considered to be statistically significant.
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