Tissue processing and iTRAQ labelling
Flash-frozen human GBM operative samples, non-malignant brain tissues (n = 6 each) and paraffin-embedded GBM tissues sections were obtained from the University of Toronto, Nervous System Tumor bank, in accordance with Research Ethics Board guidelines and consent. Six tissue samples, each of GBM and non-malignant brain (NB), were washed thrice in 1 mL of cold phosphate buffered saline (PBS, 0.1 M, pH = 7.2) and homogenized in 0.5 mL of PBS with a cocktail of protease inhibitors (1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 10 μ M leupeptin, 1 μg/mL aprotinin and 1 μM pepstatin), centrifuged and the supernatant was collected. The total protein concentration was determined using a Bradford-type colorimetric assay as described earlier [7–11]. For each set, clarified sample lysates were individually denatured, alkylated, digested with trypsin and labeled with iTRAQ labels. The labeled samples were then pooled to form three 4-plex iTRAQ sets (see Additional file 1:Table S1). Each iTRAQ set was processed by offline two-dimensional LC-MS/MS analysis as described earlier [7–11].
Mass spectrometry and data analysis
Tandem MS analysis was performed on a QSTAR Pulsar (Applied Biosystems Inc/ MDS Analytical) instrument in information dependent acquisition (IDA) mode using Analyst QS version 1.1 (Applied Biosystems Inc) as described [7–11]. Data from each set of fractions were collectively searched against a database downloaded from Uniprot (download date: 2nd June 2010) that contained 34950 protein sequences, using ProteinPilot version 4.0 (Applied Biosystems). Besides matching spectra with sequences, ProteinPilot also features a grouping function that minimizes the redundancy between the proteins reported. Proteins reported herein are, therefore, only those for which an unshared peptide or group of peptides were detected. To provide a measure of confidence in the proteins reported, the database against which the mass spectrometric data was searched included a decoy database generated by reversing the protein sequences in the original database and concatenating this reverse database to the original database. Any proteins matching the reverse sequence are obviously false, thereby providing a basis for calculation of the false discovery rate (FDR). This FDR calculation was described in Tang et al., .
Relative quantification of proteins, which was simultaneously performed by ProteinPilot, was based on the areas of the iTRAQ signature ion peaks. Only peptides not shared with other reported protein (or group) contribute to the overall ratios reported for the protein. The overall protein ratios reported is a weighted average of the ratios of the contributing unique peptides, where the weighting factor is determined by the % error of the individual peptide ratios. Further, the reported ratios were normalized using a factor, termed the applied bias that is calculated based on the assumption that the majority of the proteins being compared between the samples in a set are expressed at similar levels .
To minimize redundancy between proteins reported in the three individual iTRAQ sets and to ensure consistency of reported isoforms from one set to the next, the results of the three sets were aligned using an Excel based Protein Alignment Template (an early version of which was kindly provided by Dr. Sean Seymour, AB SCIEX) . A master list of all the proteins identified in this study was first generated by performing a search on the combined data from all three iTRAQ sets and duplicate runs using ProteinPilot. The Protein summaries from the ProteinPilot results for the individual sets were then imported into the template and the proteins were collated using the master list as the reference. The complete list and the individual ratios for each protein in each set are shown in Additional file 2: Table S2.
Equal amounts of whole cell lysates from GBM (n = 3) and non-malignant brain tissues (n = 2) as used in iTRAQ analysis were subjected to Western blotting . Briefly, equal amounts of proteins (50 μg) obtained from GBM, non-malignant brain tissues and glioma cells were resolved on sodium dodecyl sulphate - polyacrylamide gels (SDS-PAGE). The proteins were then electro-transferred onto nitrocellulose membranes (BioRad, Hercules, CA). After blocking with 5% non-fat powdered milk in Tris-buffered saline (TBS, 0.1 M, pH = 7.4), blots were incubated with mouse monoclonal anti-moesin (cat no. ab3196) / anti-γ-enolase (cat no. sc-376375) / anti-β-actin (cat no. ab123020), rabbit monoclonal anti-CD44 (cat no. ab51037) / rabbit polyclonal 14-3-3ζ (cat no. sc1019) / anti-S100A11 (cat no. sc-98427) antibody at 4°C overnight. Membranes were incubated with secondary antibody, HRP-conjugated / rabbit / mouse anti-IgG (BioRad, CA), diluted at an appropriate dilution in 1% bovine serum albumin (BSA), for 2 h at room temperature. After each step, blots were washed three times with Tween (0.1%) -Tris-buffer saline (TTBS). Protein bands were detected by the enhanced chemiluminescence method (GE Health Care) on Kodak Hyperfilm.
Immunohistochemistry of moesin and CD44 in GBM tissues
Immunohistochemistry for moesin and CD44 were carried out in independent set of paraffin embedded sections of GBM in tissue microarray format (TMA) as described earlier . In brief, the sections were deparaffinized in xylene, hydrated in gradient alcohol, and pre-treated in a microwave oven for 15 min at maximum power in citrate buffer (0.01 M, pH = 6.0, 0.05% Tween-20) for antigen retrieval. The sections were incubated with hydrogen peroxide (0.3% v/v) in phosphate buffered saline (PBS, 0.1 M, pH = 7.2) for 15 min to quench the endogenous peroxidase activity, followed by blocking with 5% BSA to preclude non-specific binding. Thereafter, the slides were incubated with mouse monoclonal anti-moesin antibody / rabbit monoclonal anti-CD44 for 16 h at 4°C. The primary antibody was detected using the streptavidin-biotin complex with the Dako LSAB plus kit (Dako Cytomation, Glostrup, Denmark) and diaminobenzidine as chromogen [10, 13, 37]. All procedures were carried out at room temperature unless otherwise specified. Slides were washed three times using PBS containing 0.025% Triton-X-100, after every step. Finally, the sections were counterstained with Mayer’s hematoxylin and mounted with DPX mountant. In the negative control tissue sections, the primary antibody was replaced by isotype specific non-immune mouse IgG. The sections were evaluated by light microscopic examination.
Cell culture, treatment with HA and siRNA transfections
GBM cells (U87 and U373) and normal human astrocytes (NHA) immortalized with human telomerase construct (hTERT) were a kind gift from Dr. Abhijit Guha, The Hospital for Sick Children, University of Toronto, Toronto, Ontario. Cells were grown in monolayer cultures in Dulbecco’s modified eagle medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% FBS, 1 mM L-glutamine, 1 mM minimum essential medium (MEM), 100 μg/mL streptomycin and 100 U/mL penicillin in a humidified incubator (5% carbon-dioxide, 95% air) at 37°C as described earlier [11, 36]. Both the GBM cell lines and NHA cells were treated with HA (Sigma St. Louis, MO) at dose range (50 - 200 μg/mL) for 6 - 48 h. For all experiments, HA was suspended in the growth medium, i.e. DMEM only without FBS. Effect of treatment on cell migration, proliferation, cell cycle and expression of moesin, CD44 was determined as described below. Pre-validated siRNA targeting moesin was obtained from Ambion (Life Technologies, CA). Negative control siRNA showing no homology to any of the known mammalian gene (i.e., having a scrambled sequence) and Cy3-labeled control siRNA for determining transfection efficiency were also obtained from Ambion (Life Technologies, CA). Transient transfection of siRNAs was performed using Lipofectamine 2000 and cells were collected after 48 h post-transfection for further Western blot analyses as described earlier [11, 36].
Wound-healing and cell migration assays
For wound-healing assays, 1×106 GBM cells (U87 / U373) or NHA cells were plated in 6-well plates for 24 h. Cells were either left untreated (no treatment controls), treated with HA (50 - 100 μg/mL) suspended in DMEM only or transfected with siRNA targeting moesin (200 nM, 24 - 72 h). Further, to determine the role of moesin downstream of HA - CD44 induced cell migration, glioma cells were transfected with moesin siRNA followed by treatment HA (100 μg/mL) for another 48 h. Similar-sized wounds were created in monolayer cells by scraping a gap using a micropipette tip either before treatment with HA or 24 h after siRNA transfections. After removing cell debris by rinsing with phosphate-buffered saline, fresh medium was added, and the cells started migrating from the edge of the wound and repopulated the gap area. Cells were then observed for ‘wound closure’ under a light microscope after 6 – 48 h and the number of cells were counted in the wound for quantitative analysis among treated cells and no treatment controls.
Further these results were verified by carrying out migration assays performed using gelatin-coated polycarbonate filters (pore size, 8 μm) on transwells separating the upper and lower chamber of 24-well plates (BD BioSciences). Chemotaxis medium (DMEM with 0.5% BSA and 10 mM HEPES) was added to the bottom chamber. siRNA transfected, HA-treated or no transfection control cells (1 × 105 / well) suspended in DMEM containing either HA (100 μg/mL) or DMEM only used as no treatment controls (NTC) were placed in the upper chamber. The plates were incubated at 37°C overnight. Viable cells in the lower chamber were collected and counted .
Cell proliferation assay
Glioma cells (U87 / U373) were plated in triplicates in 96 - well plates in complete medium (i.e. DMEM containing 10% FBS). The cells were cultured overnight and then either treated with HA (50 – 200 μg/mL) in DMEM only, for 24 - 48 h to determine dose- and time-dependent effect on cell proliferation. Cell proliferation was measured by adding MTT at 37°C for 3 - 4 h. The formazan crystals were dissolved in 100 μl of dimethylsulphoxide (DMSO) and the optical density (OD) was measured at a wavelength of 570 nm as described earlier .
Cell-cycle analysis using flow cytometry
HA-treated (100 μg/mL) or untreated no treatment control glioma cells (U87 / U373) were collected and centrifuged to collect non-adherent cells. Adherent cells were washed with phosphate buffered saline (PBS, pH = 7.4) and trypsinized. Both non-adherent and adherent cell populations were pooled for further analysis. Cells were fixed in 70% ethanol and resuspended in buffer containing PBS (pH = 7.4), EDTA (0.5 M, pH = 8.0), Triton X-100 (0.05%), RNAse A (50 μg/ml) and propidium iodide (PI, 100 μg/ml) before flow cytometry analysis. The PI-labeled cells were analyzed using a BD Canto flow cytometer and the output thus obtained was analyzed using the BD Cell Quest Pro software as described earlier . Cells were gated to exclude cell debris and cell clumps.
Co-immunoprecipitation (co-IP) assays
Co-immunoprecipitation assays to determine CD44 - moesin interactions were carried out using specific antibodies (CD44 / moesin) and analyzed by Western blotting as described earlier . Briefly, GBM cells (U87 / U373) treated with either HA (100 μg/mL, 48 h) or (TNF-α, 10 nM, 24 h) and no treatment control cells were rinsed in ice-cold PBS and lysed in IP lysis buffer . Lysates were incubated on ice for 30 min. and cell debris was removed by centrifugation. Lysates were pre-cleared by adding 20 μL of Protein A agarose (Santa Cruz Biotech., Santa Cruz, CA), followed by overnight incubation with specific antibodies (CD44 / moesin) on a rocker at 4°C. Immunocomplexes were pulled down by incubating with Protein A-agarose for 2 h at 4°C, followed by washing with 1X ice-cold lysis buffer to eliminate non-specific interactions. Protein A-agrose-bound immunocomplexes were then resuspended in Laemelli sample buffer (1X), boiled for 5 min. and analyzed by Western blotting. In addition, glioma cells treated with tumor necrosis factor-α (TNF-α) were used as a positive control for co-IP experiments . In negative controls, primary antibodies used for co-IP was replaced by isotype IgG controls .
Each experiment was repeated at least twice or was performed in triplicates. Significance of the difference between two measurements was determined using Students t-test; p-value < 0.05 was considered as significant [11, 36]. Numerical data are represented as the mean ± standard deviation.