Human gastric cancer cell line AGS was kindly provided by Min-Chuan Huang
(National Taiwan University, Taiwan) in 2010. These cells were grown in 10-cm culture
plates and maintained in RPMI medium with 10% fetal bovine serum, 2% sodium
bicarbonate, 2 mM L-Glutamine, and 1% penicillin, 1% streptomycin, and 1%
amphotericin at 37°C with 5% CO2 in a 95% humidified atmosphere.
3-2. Western blot analysis
Preparation of cell lysates
Total cell lysates from cultured AGS cells were prepared as below. The culture plate
was washed with PBS to remove the residual medium, and the cells were lysed
with 10X RIPA buffer diluted with ddH2O (1:9). Then, cells were scraped from culture
plate and incubated on ice for 15 minutes. Next, extracts were centrifuged at 14,000 rpm
for 10 minutes at 4℃. Finally, supernatant was removed to new eppendorf for use.
Bradford assay
The Bradford assay was used to determine the concentration of proteins. 1µl of
standard protein (BSA) of known concentration (3, 10, 15, 20µg/ml) and tested proteins
were mixed with 199µl 5X Bradford diluted with ddH2O (1:4). Samples were incubated
at room temperature for 5 minutes and the absorbance was measured at 630nm. The
concentration of each sample was calculated by comparing the absorbance with the
calibration curve.
Sample preparation
Protein samples were mixed with 5X sample buffer (1:4) (5% SDS, 20% glycerol,
0.004% bromphenol blue, 125mM Tris-HCl, 10% β-mercaptoethanol, pH 8.0) and
boiled at 100℃ for 5 minutes.
Electrophoresis and electro-transfer
Equal amount (30µg) of extracted protein was loaded into each well and ran at 120V
for about 2 hours. The transfer sandwich was assembled as follows: sponge, filter
papers, gel, PVDF membrane (Millipore, 0.2µm), filter papers. Then the transfer
sandwich was relocated to the transfer apparatus filled with transfer buffer (25Mm Tris,
192mM glycine, 20% methanol.). Protein samples were transferred to PVDF membrane
at 90V for 90 minutes.
Blocking, antibody incubation and detection
The membrane was incubated with 5% BSA in TBST (50mM Tris, 150mM NaCl,
0.05% Tween 20, pH 7.5) at room temperature for 1 hours followed by incubated with
primary antibody against GALNT2 (1:1000, Sigma), EGFR (1:1000, Cell signaling
technology), phospho-EGFR (Try1068) (1:1000, Cell signaling technology), Akt
(1:1000, abcam), phospho-Akt (1:1000, abcam), ERK (1:1000, Cell signaling
technology), phospho-ERK (1:1000, abcam) and GAPDH (1:1000,Novus Biologicals)
diluted in 5%BSA/TBST overnight at 4℃. Next day, the membrane was washed 3 times
with TBST, and then incubated with mouse IgG antibody (HRP) (1:1000, GeneTex) and
rabbit IgG antibody (HRP) (1:1000, GeneTex) at RT for 1 hour. The membrane was
washed 3 times with TBST and developed with Luminata Crescendo Western HRP
Substrate (Millipore).
3-3. Immunohistochemistry
Paraffin sectioning
Paraffin-embedded tissues were sectioned at a thickness of 5 µm. Tissue sections
were stretched in 36.5°C water bath and mounted on slides coated with
3-Aminipropyltriethoxysilane (Sigma). The slides were then placed to dry at 40°C
De-paraffinization
The slides were incubated at 60°C for 30 min. Tissues were deparaffinized in 2
changes of xylene, 10 minutes each, and then rehydrated in 2 changes of 100% ethanol
for 3 minutes each, 90%, 80% and 70% ethanol for 3 minutes respectively. After the
slides were immersed in the above sequence, they were washed twice with PBS for 5
minutes each.
Antigen retrieval
The slides were incubated in sodium citrate buffer (10mM Sodium Citrate, 0.05%
Tween 20, pH 6.0) and heated using Microwave Vacuum Histoprocessor RHS-1
(Milestone) with program GPR100C (20 slides – 250mL). Then the slides were cooling
by running tap water for 20 minutes.
Staining
After antigen retrieval, the slides were washed 3 times in TBS solution then
incubated with 0.3% H2O2 for 10 minutes to block endogenous peroxidase activity
followed by PBS wash for 3 times. Then applying 5% (w/v) BSA/TBS to block
nonspecific binding for 1 hour. The sections were then incubated with anti-GALNT2
polyclonal antibody (1:200, Sigma) and phospho-EGF Receptor (Tyr1068) (1H12)
Mouse mAb (1:250, Cell signaling technology) diluted with 5% BSA/TBS overnight at
4°C. Signals were detected employing UltraVision Quanto Detection System HRP
(Thermo) and visualized by DAB quanto (Thermo). All sections were counterstained
with hematoxylin for 30 seconds and rinsed in running tap water for 2 minutes.
Dehydration and mounting
The tissue slides were dehydrated through 70%, 80% ,90% and 100% ethanol for
30 seconds respectively, and then were immersed in 2 changes of xylene, 30 seconds
each. After the slides were dehydrated in the above sequence, they were mounted with
Micromount Mounting Medium (Leica).
3-4. IHC evaluation
Two pathologists independently quantified staining. Every tumor was given a score
according to the intensity of staining (no staining = 0, weak staining =1, moderate
staining = 2, strong staining = 3) and percentage of stained cells (0% = 0, 1–10% = 1,
11–50% = 2, 50-80% = 3, >80%=4). The score gives a range of 0–12 as the product of
multiplication between stained cells percentage score (0–4) and staining intensity score
(0–3). Scoring was performed for four random distinct fields per slide, and then 4 scores
were averaged. IHC score between 4-12 was defined as phospho-EGFR positive and
score between 0-4 was defined as phospho-EGFR negative .
3-5. Real-time reverse transcription PCR (RT-PCR)
RNA extraction
The total RNA was isolated using Trizol reagent (Invitrogen, Life Technologies).
The isolation procedure was as below. The culture plates were washed three times with
PBS and cells were extracted with 1ml Trizol Reagent. Then, cells were scraped from
culture plates and incubated at room temperature for 15 minutes. Next, the homogenized
samples were mixed with 0.2ml of chloroform and were incubated at RT for 3 minutes.
Then the homogenized samples were centrifuged at 12,000g for 15 minutes at 4°C and
the aqueous phase was placed into a new eppendorf. The RNA was precipitated by
adding 0.5ml isopropanol to the aqueous phase and the mixtures were incubated at RT
for 10 min followed by centrifugation at 12,000g for 10 mins at 4°C. After the
supernatant was removed, the RNA pellet was wash twice with 1ml of 75% ethanol and
air dried. Finally, RNA was resuspended in RNase-free water.
Reverse transcription
The cDNA was synthesized with the High Capacity cDNA Reverse Transcription
Kits (Applied bio-system). 2µg RNA was mixed with RT master mix (2µl of 10X RT
Buffer, 0.8µl of 25X dNTP Mix (100mM), 2µl of 10X RT Random Primers, 1µl of
MultiScribe™ Reverse Transcriptase, 1µl of RNase Inhibitor, 3.2µl of Nuclease-free H2O.) and
then DEPC water was added to bring the total volume to 20µl.
The thermal cycler condition was programmed as followed: 25℃/10 min, 37℃/120
min, 85℃/5 min, and 4℃/∞, and then reverse transcription was performed.
Real-time PCR
The cDNA was subjected to real-time PCR using quantitative PCR System
Mx3000P (Stratagene). Primers for GALNT2 were 5-AAGGAGAAGTCGGTGA
AGCA-3 and 5-TTGAGCGTGAACTTCCACTG-3. Primers for GAPDH were
5-ACAGTCAGCCGCATCTTCTT-3 and 5-GACAAGCTTCCCGTTCTCAG-3.
Relative quantity of mRNA expression normalized to GAPDH was analyzed with
MxPro Software (Stratagene).
3-6. SiRNA knockdown of GALNT2 expression
In transient knockdown experiments, a siRNA oligonucleotides against GALNT2
(5-CAGCAGGGAACUAACUGCCUCGACA-3 and 5-UGUCGAGGCAGUUAGUU
CCCUGCUG)) and a non-targeting siRNA control were synthesized by Invitrogen. The
AGS cells (2 ×105 cells ) were transfected with siRNA using Lipofectamine
RNAiMAX Reagent (Invitrogen) with the final concentration of 10nM for 24hours. The
step-by-step procedure is listed as below. Serum-free RPMI-1640 (500µl) containing
siRNA was mixed with 500 µl serum-free RPMI-1640 containing 10 µl Lipofectamine
RNAiMAX Reagent. The resultant mixture was kept at room temperature for 20 min,
and then was added into the plates. The plates were incubated at 37°C for 24 h.
3-7. MTT assay
The cell viability was assessed by measuring the ability of cells to reduce
3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) to the dark blue
formazan product. AGS cells were seeded at a density of 2 × 103 cells/200µl per well.
For assessing the effect of gefitinib (ApexBio Technology), cells were incubated with
10% FBS containing DMSO (0.1%, Sigma) or gefitinib(1µM). The effect of MK2206
(AdooQ BioScience) was assessed by adding DMSO (0.1%, Sigma) or MK2206(1µM)
to the 96-wells. Then MTT solution was added to each well and incubated at 37°C for 3
hours. The solution was carefully removed followed by addition of DMSO. Absorbance
of sample was measured at 570 nm at day1, 2, 3, 4 and 5. Results were expressed as
percentage of absorbance compared to the control cells.
3-8. Transwell migration assay
The transfected cells (3 ×104) were re-suspended in 200µl of serum-free RPMI containing gefitinib and EGF(50ng/ml, Sigma) or MK2206 and added to the upper
chamber with an 8-µm pore size membrane (Corning) for assessing effect of gefitinib
and MK2206 on cell migration. 700µl of RMPI with 10%FBS was added to lower
chamber as chemo-attractant. After 24 hours, the non-migrating cells on the upper
surface of the membrane were removed by scrubbing with a cotton-tipped swab, and the
invaded cells on the lower surface of the membrane were fixed with 100% methanol and
then stained with 0.5% crystal violet (Sigma). The number of migrated cells per field
was counted under a phase contrast microscope. Four random fields were examined and
analyzed at 100x magnification.
3-9. Matrigel invasion assay
Procedures were the same as trans-well migration assay, except for the upper chamber
with an 8-µm pore size membrane (Corning) being coated with corning Matrigel Matrix
diluted with serum-free RPMI (1:4) at 37°C overnight the day before experiment.
3-10. VVA lectin pull down assay
Cell lysates (0.5 mg) were incubated with 30µl Vicia Villosa Lectin (VVA)-conjugated
agarose beads (Vector Laboratories) at 4 °C overnight. The lectin/glycoprotein
complexes were collected by centrifugation(10,000 rpm, 1min) and washed twice with
PBS. Glycoproteins were released from the complexes by boiled in 5µl of 5x sample
buffer for 5 minutes. The precipitated proteins were resolved by SDS– PAGE, then
immunoblotted to detect EGFR. EGFR of total lysates were served as internal control.
3-11. Statistic analyses
Data were represented as the mean ± SD. Statistical analyses were performed using Prism6. The Student t test and 2-way ANOVA followed by a Bonferroni post hoc test
were used to compare differences between experimental groups. Chi-square was used to analyze correlation between pairs of categorical variables. The Kaplan-Meier log-rank test was performed to estimate probabilities of progression-free survival. All statistical tests were 2-sided, and P < 0.05 was considered statistically significant.