The human papillary non-metastatic bladder cancer cell line RT112 was kindly provided by Prof. Alberto Bardelli (Institute for Cancer Research and Treatment, IRCC, Candiolo, Torino, Italy). Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 1% penicillin-streptomycin solution (10.000 units penicillin-G and 10 mg streptomycin per mL) and 1% L-glutamine solutions (2 mM). Media components were all from Sigma (Italy).
Carcinogen treatment and development of resistant cell lines
4-ABP was dissolved in DMSO (Sigma) and activated in NADPH generating system solution (0.27 mM NADP, 5 mM glucose 6-phosphate, 0.45 mM MgCl2, 0.45 mM KCl and 200 mM Tris-HCl, Sigma) containing 0.4 mg/mL Aroclor 1254-induced S9 rat liver extract (BD Italy) for 2 h at 37°C. Cells were treated with the carcinogen for 4 h at 37°C then washed and resuspended in complete medium. Resistant cells were established by two treatments with 125 ng/mL 4-ABP, the concentration causing 99% death of RT112 cells. Treatment was repeated after cells surviving the first round had recovered exponential growth. Single-cell clones were obtained by limiting dilution. The relative resistance of isolated clones was evaluated by treating 5 × 105 cells with appropriate concentrations of the carcinogen ranging from 75 to 175 ng 4-ABP/mL. Relative resistance of the treated cultures was normalized to the plating efficiency of the untreated controls. The full development and characterization of the resistant cell lines will be reported in a forthcoming publication (F. Saletta et al., submitted).
Cell lysis and protein solubilization for 2-DE
Cells at 80% confluent were harvested by scraping, washed three times with ice-cold 10 mM PBS and pelleted. Pellets (5 × 106 cells/pellet) were shock-frozen in liquid nitrogen and stored at -80°C until further processing. Cell lysis and protein extraction was done in a solution containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% Zwittergent and a mixture of protease inhibitors (complete, mini EDTA-free cocktail, Roche). DeStreak reagent (100 mM) (GE Healthcare, Italy) was added in order to protect cysteinyl groups and prevent non-specific oxidation. The suspension was incubated for 40 minutes at 4°C on a mixing wheel. Cell debris was removed by centrifugation at 15,000 × g for 15 min, then the supernatant was aliquoted and stored at -80°C. Protein concentration was determined using the PlusOne 2-D Quant kit (GE Healthcare, Italy).
For each gel, 300 μg of total protein were dissolved to a final volume of 250 μL in the re-hydration solution (5 M urea, 2 M thiourea, 2% CHAPS, 2% Zwittergent, 100 mM DeStreak and 0.5% IPG buffer pH 3–10 linear, GE Healthcare, Italy), and then applied on immobilized pH 3–10 linear gradient strips (IPG strip, GE Healthcare, Italy). The strips were hydrated on an IPGphor apparatus (GE Healthcare, Italy) for 16 h at 30 V/h, then focused for 26 h until 50,000 Vhr. After the first-dimension run, proteins were reduced by incubating individual strips for 15 min in a solution containing 50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 60 mM dithiothreitol (DTT, GE Healthcare, Italy). Proteins were then alkylated by incubating the strips for 15 min in a similar solution, with DTT replaced by 100 mM iodoacetamide. The strips were embedded in 0.7% (w/v) agarose on the top of 1 mm-thick acrylamide gels cast at 7.5–17.5%. Proteins were separated by mass by electrophoresis at 10 mA/gel. This was done overnight, at 4°C, in a running buffer composed of 25 mM Tris, 250 mM glycine, 0.1% SDS. Gels were rinsed three times with de-ionised H2O, fixed for 1 hour in an aqueous solution with 50% methanol and 7% acetic acid, and rinsed again with de-ionised H2O. Finally, gels were stained with colloidal Coomassie Blue (Pierce) for 4–5 hours then extensively washed with de-ionised H2O .
Three replicate gels were run for each experimental condition (parental cell lines and two resistant clones).
Stained gels were scanned at 16-bit resolution (Expression 1680 Pro, Epson) and the resulting TIFF images were analysed with Progenesis Workstation software (v2005, Nonlinear Dynamics, UK). The Progenesis automatic analysis protocol for the images of the nine gels included spot detection, warping, background subtraction, average gel creation, matching and reference gel modification. Spot volumes were normalized against the total volume of all the spots in the gel. Average gels were generated by the software for spot pattern comparison. They are a statistical combination of the gels in a group, showing mean spot values with the associated error, providing information about spot variability within the gel set. An average gel was created for each experimental group by combining the three replicates. The criterion for including a spot in the average gel was that any spot must be present in all replicates. Spot editing (spot splitting corrections and match editing) was done sparingly, and only on selected complex areas of the gel.
Statistical comparisons of the individual protein abundance in the three cell clones (one-way ANOVA) and between-groups comparisons (multiple comparison test, Tukey Kramer HSD, p < 0.05) were computed using JMP v6 software (SAS Institute Inc.).
Protein identification by mass spectrometry
In-gel digestion was done as previously described . Briefly, the spots of interest were excised manually from the gel and digested with sequencing-grade modified trypsin. Aliquots of the supernatant containing tryptic peptides were directly analysed by mass spectrometry.
Liquid chromatography (reverse-phase microbore-LC)-tandem mass spectrometry (LC-MS/MS) was done as previously reported  using a Surveyor system (autosampler and MS pump) coupled to an ion-trap mass spectrometer LCQ Deca XPPlus (Thermo Finnigan) equipped with a standard electrospray source, operated in positive ion mode, with an ion sprayer voltage of 4.6 kV and capillary temperature of 220°C.
Data were acquired sequentially in MS mode (scan range of 450–2000 amu), and in data-dependent mode, recording the MS/MS spectra of the three most intense ions of each MS scan. The MS/MS spectra were acquired with an isolation width of 3.0 amu and normalized collision energy of 45%. Raw MS/MS data from each LC run were transformed into dta files using the instrument software (BioWorks, rev. 3.1 SR1), with automatic selection of individual MS/MS spectra.
Tandem mass spectra were analysed using the MS/MS search engine Phenyx version 1.9 (GenBio, Switzerland) against the UniProt_Swiss Prot database (version 50.7).
The search was enzymatically constrained for trypsin, and allowed for one missed cleavage site. Further search parameters were: no restriction on molecular weight (MW) and isoelectric point; taxonomy: Homo sapiens; fixed modification: carbamidomethylation of cysteine; variable modification: oxidation of methionine.
A summary table is available (see Additional file 2) that concisely restates the main submission parameters including algorithm, scoring models, thresholds. All information concerning peptide identification is available in Additional file 3, derived from the Phenyx Database/AC/Peptide view results page.
The electrophoretic pattern of proteins related to apoptosis (Bax, Bcl-2, caspase-3) and mitotic checkpoints (mitotic arrest deficient 2, MAD2) was investigated by Western blot analysis. Cells extracts were prepared by lysing cells in the lysis buffer (Triton X-100, 1 M Tris, 5 M NaCl) in the presence of aprotinin, leupeptine and PMSF as protease inhibitors, for 30 minutes on ice. Insoluble materials was pelletted at 13,000 × g for 10 minutes at 4°C and the protein concentration was determined using a Biorad assay kit (BioRad, Milan, Italy).
Total cellular proteins (30 μg protein/lane) were separated on SDS-10% polyacrylamide resolving gels using the Mini Protean II electrophoresis system at 100 V, for 2 hours (Bio-Rad, Milan, Italy). Proteins were transferred to nitrocellulose transfer membrane (Whatman, UK) using the transfer buffer (50 mM Tris, 100 mM glycine, SDS 0.01%, 20% methanol) and the Bio-Rad Trans-blot system (55 V, 2 h).
Blots were rinsed with TBS-T buffer (10 mM Tris-HCl pH 8, 150 mM HCl, 0.05% v/v Tween-20) and blocked in TBS-T, 5% w/v non-fat dried milk (Nestlé, Italy) for 2 h.
After overnight incubation with primary antibody diluted 1:300 in TBS-T, 5% non-fat dried milk, (rabbit polyclonal antibodies: β-tubulin H-235, caspase-3 H-277, MAD2 FL-205, Bcl-2 N-19, Bax N-20; mouse monoclonal antibody caspase-3 E-8; Santa Cruz Biotechnology, USA) blots were washed with TBS-T and incubated with secondary antibody at 1:1000 for 2 h. Peroxidase-conjugated anti-mouse or anti-rabbit IgG HRP (Santa Cruz, Biotechnology, USA) were used as secondary antibodies.
Blots were revealed using enhanced chemiluminescence (ECL) (GE, Milan, Italy) and scanned as 16-bit images (Expression 1680 Pro, Epson). The resulting TIFF images were analysed using the Progenesis software (v2005, Nonlinear Dynamics, U.K, Nonlinear Dynamics, UK). Expression data were normalized relative to β-tubulin.