Cell culture and EGF treatment
NPC cell line CNE2 cells were cultured to 60-70% confluency in DMEM medium supplemented with 10% fetal bovine serum (Invitrogen) at 37°C, serum-starved for 24 h, and then were stimulated with 30 ng/mL EGF (Sigma) or mock-treated as a control. In EGFR blocking experiments, cells were pretreated with 1 μm EGFR tyrosine kinase inhibitor PD153035 (Calbiochem), and followed by incubation with EGF.
A phosphoprotein purification kit (BD Biosciences) was applied to enrich phosphoproteins from EGF-stimulated or unstimulated CNE2 cells according to the manufacturer's instructions. To validate the efficacy of phosphoprotein enrichment, 40 μg of proteins from total cellular lysate, elution fraction containing the highly-concentrated and purified phosphoproteins, and flow-through fraction were separated by 6% SDS-PAGE, followed by Western blotting using anti-phosphotyrosine antibody (4G10, Upstate). The concentration of the phosphoproteins was determined using a 2-D Quantification Kit (Amersham Biosciences).
Phosphoproteins from the elution fractions were precipitated using chloroform-methanol as described by Wessel and Flugge , resolubilized in 2D-DIGE sample buffer (30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, pH 8.5), and adjusted to pH 8.5. Equal amount phosphoproteins from six samples (three biological repeats) were pooled together as the internal standard. Three EGF-stimulated samples and three EGF-unstimulated samples were randomly labeled with Cy3 or Cy5, while internal standards were labeled with Cy2, using 200 pmol fluorochrome/25 μg protein (Amersham Biosciences). Labeling reactions were performed on ice in the dark for 30 min, and then quenched by the addition of 1 μL 10 mM lysine (Sigma) for 10 min.
Cy3- and Cy5-labelled samples (25 μg) from each pair of EGF-treated and untreated cells were combined before mixing with 25 μg Cy2-labelled internal standards. An equal volume of 2 × sample buffer (8 M urea, 130 mM DTT) was added to the sample and the total volume was made up to 450 μL with rehydration buffer (8 M urea, 4% CHAPS, 1% Biolyte, pH 4-7, 13 mM DTT). The samples were applied to IPG strips [pH 4-7; nonlinear (NL), 24 cm] and focused on an IPGphor (Amersham Biosciences). The focused IPG strips were equilibrated, and then were transferred to the tops of 12.5% polyacrylamide gels and run for about 7 h, using low-fluorescence glass plates on an Ettan DALT II system (Amersham Biosciences). All electrophoresis procedures were performed in the dark. The biological triplicate EGF-stimulated and unstimulated cells and the internal standard were run on three gels as analytic gels. In addition, we performed another strip in parallel as a preparative gel for spot pickings, as described above, except that the IPG strip was loaded with 1000 μg proteins and the gel was stained with Coomassie brilliant blue. After SDS-PAGE, the three analytic gels were scanned on a Typhoon 9410 scanner (GE Healthcare) at appropriate excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm) and Cy5 (633/670 nm), to generate nine protein spot maps.
Images were cropped using ImageQuant TL 2005 software (GE Healthcare) from 2D-DIGE gels, and analyzed using DeCyder 6.5 software (GE Healthcare) according to the manufacturer's recommendations. The DeCyder differential in-gel analysis (DIA) module was used for pairwise comparisons of each EGF-stimulated and unstimulated cell sample to the internal standard in each gel. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all nine protein-spot maps and, using the Cy3: Cy2 and Cy5: Cy2 DIA ratios, to calculate average abundance changes and paired Student's t -test p values for the variance of these ratios for each protein pair across all samples. The differential protein spots (ratio ≥ 1 .5, p ≤ 0 .05) that altered consistently in all nine protein-spot maps were selected for identification.
Protein identification by MS
All the differential phosphoprotein spots were excised from stained preparative gels using punch, destained, and in-gel trypsin digestion was performed as previously described by us . Briefly, the gel spots were destained with 100 mM NH4HCO3 in 50% acetonitrile, dried in a vacuum centrifuge, and incubated in the digestion solution (40 mM NH4HCO3, 9% acetonitrile, and 20 μg/mL proteomics grade trypsin) at 37°C for 14-16 h. The resulted peptides were extracted with 50% acetonitrile/2.5% TFA, purified with ZipTip C18 column (Millipore) and mixed with CCA matrix solution[4-hydroxy-α-cyanocinnamic acid (HCCA; Sigma) in 30% ACN/0.1% TFA] followed by analysis with Voyager System DE-STR 4307 MALDI-TOF Mass Spectrometer (ABI) to obtain the peptide mass fingerprint (PMF). The standard peptide mixture was analyzed at the same time to correct the machine. The parameters of MALDI-TOF were set up as follows: positive ionreflector mode, accelerating voltage 20 kV, grid voltage 64.5%, mirror voltage ratio 1.12, N2 laser wavelength 337 nm, pulse width 3 ns, the number of laser shots 50, acquisition mass range 500-3000 Da, delay 100 ns, and vacuum degree 4 × 10-7 Torr.
In peptide mass fingerprint map database searching, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss-Prot database were performed using the Mascot search engine http://www.matrixscience.com/ with a mass tolerance of ± 50 p.p.m. Protein scores of ≥ 56 (threshold) indicate identity or extensive homology (P < 0.05) and were considered significant.
To do phosphorylation site prediction of the identified proteins, we used on-line PhosphoSitePlus™ system biology resource http://www.phosphosite.org/ and the Phospho.ELM database http://phospho.elm.eu.org/index.html for predicting the presence of the phosphorylation modification sites , and PubMed database searching http://www.ncbi.nlm.nih.gov for comparing with the phosphorylated proteins reported in the literature. In addition, KEGG pathway analysis of the identified proteins was done in DAVID bioinformatics resources [18, 19].
Validation of EGFR signaling phosphoproteins by IP-Western blotting
Cells were lysed in the lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Ttiton X-100, 1% NP-40 supplemented with phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich) at 4°C, and subsequently centrifuged at 12000 g for 30 min at 4°C. Total cellular proteins were immunoprecipitated with protein G Sepharose (Amersham Biosciences), and anti-target protein antibody overnight at 4°C. Immunocomplexes were used for Western blotting. Briefly, proteins were separated by 7% SDS-PAGE, and transferred to a PVDF membrane. Blots were blocked with 3% BSA for 1 h at room temperature, and then incubated with primary antibody, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. The signal was visualized using ECL detection reagent.
The cells were transfected with GSTP1 siRNA or control siRNA (Santa Cruz Biotechnology) according to the siRNA transfection protocol provided by the manufacturer. Briefly, the day before transfection, CNE2 cells were plated into 6-well plates at the density of 105 cells/mL in DMEM medium containing 10% FBS (Invitrogen). When the cells were 60-80% confluent, they were transfected with 10 nmol/L of GSTP1 siRNA or control siRNA in serum-free DMEM medium using Lipofectamine 2000 reagent (Invitrogen). 4 h after the beginning of the transfection, the medium was replaced with DMEM medium containing 10% FCS, and continued to culture the cells for additional 44 h, and then GSTP1 expression level was determined by Western blotting.
Flow cytometry analysis of apoptotic cells after treatment of paclitaxel and EGF
At the end of the transfection, the cells were incubated with 30 nM paclitaxel and 30 ng/mL EGF for 48 h, and cell apoptosis was examined by flow cytometry as previously described by us . Briefly, cells were harvested, fixed with ice-cold 70% ethanol in PBS at -20°C for 1 h and then centrifuged at 1 500 rpm for 5 min. The pellets were incubated with 0.5% Triton X-100 (Sigma) and 0.05% RNase (Sigma) in 1 mL PBS at 37°C for 30 min, and then centrifuged at 1 500 rpm for 5 min. The cell pellets were incubated with 40 μg/mL propidium iodide (Sigma) in 1 mL PBS at room temperature for 30 min. Samples were immediately analyzed by a FACScan flow cytometry (Becton Dickinson). Apoptosis was evaluated based on the proportion of sub-G1 hypodiploid cells. Three independent experiments were done.
Analysis of cell viability by MTT after treatment of paclitaxel and EGF
At the end of the transfection, the cells were incubated with the different concentrations of paclitaxel and 30 ng/mL EGF for 48 h, and cell viability was examined using MTT assay as previously described by us . Briefly, 20 μl of 5 mg/mL MTT (Sigma) was added to each well, and the medium was removed after 4 h of incubation. 150 μL DMSO (Sigma) was added to each well for 10 min at room temperature. The absorbance of each well was read with a Bio-Tek Instruments EL310 Microplate Autoreader at 490 nm. Three independent experiments were done.
To construct EGFR signaling network (biological interaction network of the proteins) of identified phosphoproteins, functional and pathway analyses were performed using Pathway Studio 5.0 software, a tool for the description of networks and signaling pathways .