Supplementary Methods and Data
Summary
Supplementary methods are including the detail methods of SELDI-TOF MS analysis, Isolation and
identification of the target proteins, Reverse transcription-polymerase chain reaction (RT-PCR),
Quantitative RT-PCR, Western blot analysis and Small interfering RNA (siRNA) transfection.
Supplementary data is including the cell invasion assay. These supplementary data must help reader’s
understanding of this article.
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Methods
SELDI-TOF MS analysis
To discover the candidate protein, an aliquot of the stored 20-paired pre- and postoperative serum
samples was used for SELDI-TOF MS analysis with a weak cationic exchanger 2 (WCX2) (Ciphergen
Biosystems, Fremont, CA, USA). The profiling was done twice, under 4 sets of conditions, using urea
or ampholine buffer at a pH of 4.5 or 6.5. To 10 μL of each serum sample, 10 μL of a solution
containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, and 1% DTT in phosphate buffered saline (PBS)
of pH 8 (urea buffer) were added; and to 1 μL of each serum sample, 9 μL of a solution containing 7
mol/L urea, 2 mol/L thiourea, 4% CHAPS, 1% DTT, and 2% ampholites of pH 7 (ampholine buffer)
were added. The binding/washing buffer containing 50 mM sodium phosphate (pH 6.5) or 50 mM
sodium acetate (pH 4.5) was added up to 100 μL to the diluted samples, and 100 μL of each diluted
sample were applied to each spot on the WCX2 arrays. After the samples were left at room temperature
for 20 minutes, each array was washed 3 times with binding/washing buffer for 5 minutes, and twice with
distilled water (DW). The arrays were air-dried, and a saturated solution of Sinapinic acid (SPA)
(Ciphergen) in 50% v/v acetonitrile and 0.5% v/v trifluoroacetic acid was added twice to each spot as a
matrix. Each analysis was performed in duplicate. TOF mass spectra were generated using the
Ciphergen Protein Biology System II by averaging 60 laser shots with an intensity of 230 and a detector
sensitivity of 5. Peak detection was performed using ProteinChip Software 3.1 (Ciphergen). All
spectra were compiled, and qualified mass peaks with mass-to-charge ratios (m/z) between 3,000 and
30,000 were optimally auto-detected. Peak clusters were completed using the second pass peak section
(s/n ratio 0.5, within a 0.3% mass window), and estimated peaks were added. The relative peak
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intensities, normalized to a total ion current of m/z, were expressed as arbitrary units.
Isolation and identification of the target proteins
The candidate proteins were purified, isolated, and identified as follows. The optimal pH was
determined by stepwise analysis on WCX2 arrays, and it was found to be around 4.5, at which levels the
target peaks were highest. This was fixed as a condition for further experiments. The serum was
subjected to ion-exchange fractionation by fast protein liquid chromatography (FPLC) (FPLC Pharmacia
LKB; Amersham Pharmacia Biotech, Uppsala, Sweden). FPLC fractionations were monitored on a
hydrophilic NP20 ProteinChip array (1 μL sample per spot) with SPA matrix. The ion-exchange resin
and buffer conditions that were chosen were based on the ProteinChip affinity parameters during SELDI
analysis. After equilibration, fractionation was performed with a stepwise gradient, and the target
protein was eluted in the 435 mM sodium chloride fraction. Fractions rich in the specific protein were
collected, concentrated, and twice subjected to high performance liquid chromatography (HPLC)
(CCPM/PX-8010, TOSOH, Tokyo, Japan). First, HPLC was done with a sephasil protein C18 column
(Aquapore OD-300, Perkin Elmer). Then after passage through a C4 column (Cadenza CD-C4, Intakt)
for albumin removal, a second HPLC was performed with a C18 column (Cadenza CD-C18, Intakt)
followed by elution with a linear gradient of 0.1%-80% acetonitrile at a flow rate of 200 μL/min. HPLC
fractionations were monitored on a Gold Chip array (1 μL sample per spot) with the SPA matrix. After
purification, the target protein was identified by N-terminal amino acid sequence analysis.
For
immunodepletion assay, 3 μg of anti-human apolipoproteinC-1 monoclonal antibody (CHEMICON
International, Inc. Temecula, CA, USA) and control mouse monoclonal IgG (X0943, DAKO, Glostrup,
Denmark) were incubated with 10 μl protein A-agarose (Sigma Chemical, St. Louis, MO, USA) for 15
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minutes on ice. The pellet was washed twice with buffer containing 20 mM Hepes (pH 7.8), 25 mM
KCl, 5 mM MgCl2, 0.1 mM EDTA and 0.05% NP-40. Hundred-μl serum samples were incubated
with the appropriate pellet for 2 hours on ice. After centrifugation, 3 μl of each supernatant were
analyzed using the CM10 ProteinChip arrays.
Reverse transcription-polymerase chain reaction (RT-PCR) and Quantitative RT-PCR
PCR was done with the following primer sets; apolipoprotein C-1 (ApoC-1): forward
5’-CTCCAGTGCCTTGGATAAGC-3’, reverse 5’-TTGAGTTTCTCCTTCACTTTCTGA-3’, β-actin:
forward 5’-CTCTTCCAGCCTTCCTTCCT-3’, reverse 5’-AGCACTGTGTTGGCGTACAG-3’, and
glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH):
5’-ACCCAGAAGACTGTGGATGG-3’, reverse 5’-TTCTAGACGGCAGGTCAGGT-3’.
forward
The
RT-PCR conditions for ApoC-1 and β-actin were as follows: 95°C for 5 minutes, 37 cycles at 95°C for 15
seconds, 58°C for 15 seconds, and 72°C for 1 minute, with an extension step of 7 minutes at 72°C at the
end of the last cycle. The conditions for quantitative RT-PCR were as follows. For ApoC-1, the initial
denaturation was at 95°C for 10 minutes, followed by 45 cycles denaturation at 95°C for 10 seconds,
annealing at 58°C for 10 seconds, extension at 72°C for 7 seconds; for GAPDH the same conditions were
used. ApoC-1 mRNA levels were determined as the absolute copy number normalized against that of
GAPDH mRNA.
Western blot analysis
To extract protein from the cultured cells, the cells were lysed in buffer (250 mM Tris HCl, 40% glycerol,
5% SDS, 5% BPB, and 5% β-ME). The lysate was incubated for 5 minutes at 100°C followed by
centrifugation (15,000 rpm) for 15 minutes at 4°C, and the supernatant was subjected to Western blot
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analysis. To analyze the protein secreted from the cultured cells, the supernatant was collected and
centrifuged for 10 minutes at 4°C; 10 μl of the supernatant was subjected to Western blot analysis.
Frozen tissue samples were solubilized in lysis buffer (7 M Urea, 2 M thiourea, 4%
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 1% DTT, 2% pharmalyte (Amersham
Pharmacia Biotech, Buckinghamshire, UK), and 30 mM Tris, containing protease inhibitor) by
homogenization followed by ultracentrifugation at 55,000 rpm for 1 hour at 4°C. The supernatant was
subjected to Western blot analysis. Twenty μg of each protein were separated by electrophoresis on
10%-20% gradient gels (PerfectNT Gel, DRC, Tokyo, Japan) and transferred to polyvinylidene fluoride
membranes (Millipore, Bedford, MA, USA). After blocking with 0.5% skim milk in PBS, the
membranes were reacted with mouse anti-human ApoC-1 monoclonal antibody (CHEMICON
International) or with goat anti β-actin (Santa Cruz, CA, USA) antibody diluted 1:500 in blocking buffer
followed by reaction with goat anti-mouse IgG horseradish peroxidase (Bio-Rad) diluted 1:1000, or
rabbit anti-goat IgG horseradish peroxidase (Cappel, West Chester, PA, USA) diluted 1:500. Antigens
on the membrane were detected with enhanced chemiluminescence detection reagents (Amersham
Pharmacia Biotech).
Small interfering RNA (siRNA) transfection
Twenty-four hours before transfection, a total of 20 x 104 cells were plated in 6-well plates, cultured in
the appropriate medium containing fetal bovine serum (FBS) without antibiotics. One μL siRNA
oligomer and 3 μL LipofectamineTM 2000 reagent (incubated for 5 minutes at room temperature) were
diluted in 250 μL Opti-MEMⓇ I Reduced Serum Medium (GIBCO), respectively. The diluted
oligomer was mixed gently with the diluted transfection reagent. After incubation for 20 minutes at the
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room temperature, 1000 μL the siRNA oligomer-transfection reagent complexes diluted in Opti-MEMⓇ
I Reduced Serum Medium (at 20 nM final concentration) were added to each well containing cells
washed with PBS beforehand. After incubation for 4 hours in a humidified atmosphere containing 5%
CO2 at 37° C, FBS were added to each well with final concentration of 10 % in medium. The cells
were incubated in a humidified atmosphere containing 5% CO2 at 37° C for 24-48 hours until use.
Data
Inhibition of ApoC-1 expression did not affect invasion ability of these two pancreatic cancer cell lines.
MIA PaCa II
AsPC-1
120
Invasion cells (% of control)
Invasion cells (% of control)
120
100
80
60
40
20
0
100
80
60
40
20
0
GL2siRNA
ApoC-1siRNA1 ApoC-1siRNA2
GL2siRNA
ApoC-1siRNA1
ApoC-1siRNA2
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