Plasmids, cells, transfection and reagents
The gene targeting vector, pAAV-puro, was a gift of Dr. Guang bin Luo (Case Western Reserve University, USA). HCT116 (American Type Culture Collection, Rockville, MD, USA) and its derivatives were grown in 10% FBS and 1% penicillin-streptomycin in McCoy’s 5A modified media and maintained at 37°C in 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol and colonies were selected with the use of 1 μg/ml puromycin. The anti-p53 (sc-126) and anti-gankyrin (sc-8991) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the antibody against PIG3 (BS2085) and ANAX2 (BS3553) were purchased from Bioworld Technology, Inc. (Bioworld, USA). The monoclonal anti-α-tubulin (T5168) was purchased from Sigma.
Generation of gankyrin null cells
The general strategy for creating the gankyrin null line was described by Bunz et al. (2007) 9. Briefly, the 5’ and 3’ homology arms used for constructing the targeting vectors were PCR-amplified from HCT116 genomic DNA, using primers chosen from publicly available genomic sequence databases (Gene ID: 5716). The arms were cloned into vectors that contained a hygromycinythymidine kinase fusion gene that was flanked by LoxP sequences. The primers used to derive the targeting vectors and details of their construction are available from the authors upon request. Screening for homologous recombination events was performed by PCR (forward: 5’-ATGTATTCTTATCGTTACCTAGT-3’; reverse 5’-GGACGTAAACTCCTCTTCAGA-3’). After Cre-mediated LoxP excision, allele-specific primers were used for further genetic verification. The above forward primer was used with the following reverse primers: 5’-CTGTTTTGACTGGCGTAGCC-3’ for the wild-type allele and 5’-TTCTGCTTCTCTCAGAAACGG -3’ for the deleted allele. Lox recombination was mediated by transfecting the cells with a Cre expressing plasmid, pCX-Cre. All targeted clones identified by PCR were verified by Southern blotting with 10 mg of genomic DNA digested with the restriction enzyme HindIII and AatII, and then probed with a 600-bp genomic fragment lying inside the 5’ homology arm.
Cell growth curve
Equal numbers (2 × 104) of HCT116 gankyrin−/− cells or wide type cells were plated into 12-well tissue culture dishes and cell numbers were determined by counting with a hemocytometer at 0, 1, 2, 3, 4, 5 and 6 days of cell plating. Each assay was completed in triplicate.
Soft agar assay and tumorigenicity in nude mice
To assess the anchorage independency of growth, 5 × 103 cells were plated in 0.6% agar layered on top of 1.0% agar in 6-well plates, and colonies were counted after 2~3 weeks of incubation at 37°C and 5% CO2 in air. Each assay was completed in triplicate. Statistical differences between the sample means were calculated by analysis of variance (ANOVA), followed by an unpaired Student’s t-test. The results are expressed as the mean ± standard error of the mean (SEM). The animals used in this study were nude mice, 4~6 weeks old (National Center of Biomedical Analysis, Beijing). HCT116 gankyrin−/− cells and control cells (5 × 106) were implanted by subcutaneous injection into the dorsal region near the thigh of female nude mice. Tumor volume was measured at the indicated days.
Cell migration assay
The cell migration assay was performed using OrisTM Cell seeding stoppers (Platypus technologies, Madison, WI) according to the manufacturer’s protocol. Briefly, the assay utilizes OrisTM Cell seeding stoppers (made from a medical-grade silicone) to restrict cell seeding to the outer annular regions of the wells. Cells were seeded onto each well (5 × 104 cells/well) and allowed to attach for 4 h at 37°C. The stopper was subsequently removed to form an unseeded region (2 mm in diameter) at the center of each well. The plate was incubated at 37°C to permit cell migration, and the migrated cells were stained with crystal purple.
To obtain total protein lysates, 80~90% of confluent cells was washed with chilled phosphate buffered saline (PBS), and cell lysates were then prepared on ice using cool lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris, 1 mM EDTA, 1 mM EGTA, 60 mM DTT) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The sample was aliquoted and stored at −70°C until use.
2D electrophoresis and image analysis
Proteins were separated by 2-DE as described previously . Briefly, isoelectric focusing was performed with the IPGphor system (Amersham Pharmacia Biotech, Uppsala, Sweden). Several different IPG (immobilized pH gradient) strips (18 cm, pH 3~10 nonlinear, 3~5.6, 6~11, Bio-Rad Co.) were used. After active rehydration for 12 h at 30 V, the strips (18 cm, pH 3~10, nonlinear) were focused at 0.05 mA/IPG strip for 80,000 Vh at 20°C. Once the IEF was finished, the IPG strips were immediately equilibrated in 10 ml of equilibration solution (6 M urea, 30% glycerol, 2% sodium dodecyl sulfate [SDS], 50 mM Tris-Cl pH 8.8, 1% dithiothreitol [DTT]) with gentle shaking for 15 min. The strips were then treated with the same solution containing 2.5% iodoacetamide instead of DTT. SDS-PAGE was performed using 13% polyacrylamide gels without a stacking gel in the PROTEAN II cell (Bio-Rad Co.). Following SDS-PAGE, gels were stained with 0.1% (w/v) Coomassie Blue G-250 (CBB G250) in 50% methanol and 10% acetic acid, or silver-stained. Spot detection, quantification, and matching were performed with Image Master 2-D Elite Version 5.0 software according to the manufacturer’s instructions (GE Healthcare Life Sciences, Uppsala, Sweden). Proteins were subjected to further analyses when the expression levels of given protein spots changed by at least 1.5-fold. Each experiment was performed at least in triplicate.
2-DE gels were stained with silver or CBB G250. The protein spots were excised from the 2-DE gels and in-gel digested as described previously. Protein identification was repeated at least twice using spots from different gels. Protein spots were analyzed by NanoLC-HDMS MS/MS on an AcquityTM Nano UPLC system (Waters Corp., Milford, USA) and Synapt high-definition mass spectrometry (HDMS) was performed with a nanospray ion source (Waters). The HDMS was operated in a data-dependent mode with MS/MS scans (2 seconds). The voltage of non-coated capillary was set as 2300 V.
Glufibrinopeptide was used to calibrate the instrument in the MS/MS mode. Peak lists were generated using PLGS 2.2 software and automatically combined into a single pkl file for every LC-MS/MS run. The MS/MS data were acquired and processed using MassLynx V4.1 software (Micromass) and Mascot from Matrix Science in March 2011 was used to search the database using the following parameters: database, NCBInr (13366630 sequences); taxonomy, Homo sapiens (235802 sequences); enzyme, trypsin; and one missed cleavage was allowed. Carbamidomethylation was selected as a fixed modification and oxidation/phosphorylation was allowed to be variable. The peptide and fragment mass tolerances were set at 1 and 0.2 Da, respectively. Proteins with probability-based MOWSE scores that exceeded their threshold ( p < 0.05) were considered to be positively identified. If proteins were identified by a single peptide, the spectrum was validated manually. For a protein to be accepted, the assignment had to be based on four or more y- or b-series ions.
Total RNAs were isolated from 1 × 106 cells with TRIzol reagent (15596–026, Invitrogen) according to the manufacturer’s instructions. Primers used to amplify the gene fragments are available from the authors. MMLV Reverse Transcriptase (M1701, Promega, Madison, WI, USA) were used for cDNA synthesis. Transcripts were quantified by qRT-PCR on an ABI PRISM 7300 Sequence Detector (Perkin-Elmer Applied Biosystems) with TaKaRa predisigned SYBR® Premix Ex Taq™ assays and reagents (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The comparative threshold cycle method and an internal control (GAPDH) were used to normalize the expression of the target genes.