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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.

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 MSanalysis with a weak cationic exchanger 2 (WCX2) (CiphergenBiosystems, 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. To10 μL of each serum sample, 10 μL of a solutioncontaining 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, and 1% DTT in phosphate bufferedsaline (PBS) of pH 8 (urea buffer) were added; and to 1 μL of each serum sample, 9 μL of a solutioncontaining 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) or50 mM sodium acetate (pH 4.5) was added up to 100 μL to the diluted samples, and 100μL of each diluted samplewere 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 wereair-dried, and a saturated solution of Sinapinicacid (SPA) (Ciphergen) in 50% v/v acetonitrile and 0.5% v/v trifluoroaceticacid was added twice to each spot as a matrix. Each analysis was performed in duplicate. TOF massspectra were generated using the Ciphergen Protein BiologySystem II by averaging 60 laser shots with an intensity of230 and a detector sensitivity of 5. Peak detection was performed using ProteinChip Software 3.1 (Ciphergen). All spectra were compiled,and qualified mass peakswith mass-to-charge ratios (m/z) between 3,000 and30,000 were optimally auto-detected. Peak clusters were completedusing the second pass peak section (s/n ratio 0.5,within a 0.3% mass window), and estimated peaks wereadded. The relative peak intensities, normalized to a totalion current of m/z, wereexpressed 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 subjectedto ion-exchange fractionation by fast protein liquid chromatography (FPLC) (FPLC PharmaciaLKB; Amersham Pharmacia Biotech, Uppsala, Sweden). FPLC fractionations were monitored ona hydrophilic NP20 ProteinChip array (1 μL sample per spot) with SPA matrix. The ion-exchange resin and buffer conditions that were chosen werebased on the ProteinChip affinity parameters during SELDIanalysis. After equilibration, fractionation was performedwith a stepwise gradient, and the target protein was eluted in the 435 mM sodium chloride fraction. Fractions rich in the specific protein werecollected, concentrated, and twice subjected to high performance liquid chromatography (HPLC)(CCPM/PX-8010, TOSOH, Tokyo, Japan). First, HPLC was done with a sephasilprotein 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) followedby elution with a linear gradient of 0.1%-80% acetonitrile at a flow rate of 200 μL/min. HPLC fractionations were monitored ona GoldChip 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 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): forward5’-ACCCAGAAGACTGTGGATGG-3’, reverse 5’-TTCTAGACGGCAGGTCAGGT-3’. 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, and5%β-ME). The lysate was incubated for 5 minutes at100°C followed by centrifugation (15,000rpm) for 15 minutes at 4°C, and the supernatant was subjected to Western blot 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 toWestern blot analysis. Frozen tissue samples were solubilized in lysis buffer (7M Urea, 2M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 1% DTT, 2% pharmalyte(Amersham Pharmacia Biotech, Buckinghamshire, UK), and 30mM Tris, containing protease inhibitor) by homogenization followed by ultracentrifugation at 55,000rpm for 1hour 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-1monoclonal 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 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.

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