Materials
All primary and secondary antibodies, siRNA duplex and a scrambled sequence control siRNA were from Santa Cruz. LipofectamineTM RNAiMAX transfection reagents and serum-free medium were from Invitrogen. Immobiline DryStrip gels (pH 3-10 NL, 24 cm), CyDye DIGE fluor Cy2, Cy3 and Cy5 were from GE Healthcare. Matrigel basement membrane matrix was from BD Biosciences. Transwell plates and inserts (Cat. No. 3422) were from Corning. All other chemicals, unless stated otherwise, were obtained from Sigma.
Cells culture
NPC 5-8F and 6-10B cells were obtained from the Modern Analytical Testing Center of Central South University of China. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in a humidified incubator with 5% CO2 in air at 37°C. The cells were grown to 80% confluence, washed twice with D-Hank’s solution, digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetate dehydrate (EDTA), collected by centrifugation (400 × g, 10 min, 4°C). A total of ~2 × 107 cells were needed for experiments.
Preparation and characterization of mitochondria, and extraction of mitochondrial proteins
Mitochondria were isolated from 5-8F and 6-10B cells, respectively, according to the Sucrose Gradient Separation Manual (http://www.mitosciences.com) with a minor modification. Briefly, the collected cells described above were resuspended in five volumes of Mito buffers (0.25 M sucrose, 1 mM EDTA, 10 mM Tris·HCl pH 7.4, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 1 mg/ml pepstatin) and homogenized by pre-cooled Potter-Elvehjem homogenizer (1,000 rpm; around 20 up and down strokes). The homogenates were centrifuged (1,000 × g, 10 min, 4°C) to collect the supernatants. The supernatants were centrifuged (12,000 × g, 15 min, 4°C) to collect the pellets. The pellets were resuspended in 0.5 ml Mito buffers. The discontinuous sucrose gradient separation solutions were prepared in centrifuge tubes with gradient from bottom to top that is 0.5 ml of 2.0 M sucrose, 1 ml of 1.3 M sucrose, 1 ml of 1.0 M sucrose, and 0.5 ml of 0.8 M sucrose. The resuspended samples were carefully applied to the top of gradient solutions and centrifuged (80,000 × g, 2 h, 4°C; Beckman swinging bucket 55 Ti rotors). The brown belts between 1.0 M and 1.3 M sucrose layers were collected carefully with a 5-ml syringe needle immediately after centrifugation. A volume (600 μl) of methanol was added to the top of 150 μl brown sucrose solution, and mixed; then 150 μl of chloroform and 450 μl of water were added, mixed again, and centrifuged (18,000 × g, 5 min, 4°C). All organic and inorganic liquids in centrifuge tubes were poured off carefully, followed by air dry at 4°C of the white discs of proteins. The white discs of proteins were solubilized (2 h; intermittent mix) in 800 μl lysis buffer that contained 8 M urea, 2 M thiourea, 130 mM dithiothreitol (DTT) , 4% 3-[(3-cholanidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 40 mM tris base, 1 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and pH 8.5 adjusted by 50 mM NaOH, and centrifuged (18,000 × g, 2 h, 4°C). The supernatants were collected. The protein concentration of the supernatants was determined by a 2-D Quantification kit (GE Healthcare). The isolated mitochondria were characterized according to Murayama’s method [44]. Briefly, the extracted mitochondrial proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes (Semi-dry Nova blot, Pharmacia Biotech), probed with various primary antibodies against marker proteins from different cellular compartments (LDH, PCNA, COX IV, catalase, GRP 78, and cathepsin D) and corresponding secondary antibodies, and visualized by a SuperSingal West Pico ECL kit (Thermo). Preparation of isolated mitochondria for electron microscopic observation was carried out according to the related manual and observation was made by a Hitachi H 7500 transmission electron microscope.
2D-DIGE and imaging analyses
The 2D-DIGE experimental flow-chart was shown (Figure 10). Briefly, Mitochondrial proteins were labeled with fluorescent cyanine dyes following the Instruction of Amersham CyDye DIGE Fluors (minimal dyes) for Ettan DIGE Product Booklet. An amount (50 μg) of proteins were labeled with 400 pmol of dyes (dark; on ice; 30 min) and then quenched (10 min) with 1 μl of 10 mM lysine. Two sets of metastatic 5-8F cell proteins were labeled with Cy3 and the third set with Cy5. Two sets of nonmetastatic 6-10B cell proteins were labeled with Cy5 and the third set with Cy3. Each set of above sample contained 50 μg proteins. The internal standard (75 μg of metastatic 5-8F cell proteins plus 75 μg of nonmetastatic 6-10B cell proteins) was labeled with Cy2. One set (50 μg) of Cy3-labeled proteins was combined with one set (50 μg) of Cy5-labeled proteins and combined again with one third (50 μg) of Cy2-labeled internal standard proteins. The combined sample was added in equal volume of 2 × sample buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 2% Pharmalyte 3-10), and then supplied with rehydration buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 1% pharmalyte 3-10) to the total volume of 450 μl. The mixed sample (450 μl) was applied to an immobiline DryStrip gel; and three analytic gels and one preparative gel were simultaneously focused on an IPGphor Unit (GH Healthcare) with a total of 80,000 Vhr. The focused proteins in the immobiline DryStrips were equilibrated in a solution containing 65 mM DTT (15 min), and in another solution containing 135 mM iodacetamide (15 min). The equilibrated proteins in the immobiline DryStrip were separated by 12.5% SDS-PAGE gels in an Ettan DALT II system (GE Healthcare). After electrophoresis, each gel was scanned on a Typhoon 9410 scanner (GE Healthcare) at appropriate wavelengths that were specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). In total 9 protein-spot image maps were generated. Each gel-spot density was normalized by the total density in each protein-spot image map. The DeCyder differential in-gel analysis (DIA) module was used for pair-wise comparison of each 5-8F and 6-10B protein to the internal standard in each gel. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all 9 protein-spot image maps. The normalized Cy3/normalized Cy2 and normalized Cy5/normalized Cy2 DIA ratios were used to calculate average abundance changes. The average of three DIA ratios from metastatic cell 5-8F mitochondria represents the relative expression level of a protein in metastatic cell mitochondria, and the average of three DIA ratios from nonmetastatic cell 6-10B mitochondria represents the relative expression level of a protein in nonmetastatic cell mitochondria. The paired Student’s t-test p-values were used for the variance of these ratios for each protein pair across all samples (all 9 protein-spot image maps) to determine each DEP-contained gel-spot. Each DEP-contained gel-spot was excised for protein identification.
MS-identification of mitochondrial DEPs
The 2D gel spots that contained DEPs were excised from the preparative gels. The proteins in the gel-spot were destained, reducted, alkylated, and digested in gels with trypsin [45]. For PMF analysis, the tryptic peptides were eluted from ZipTips with 80% acetonitrile/0.1% trifluoroacetic acid, and were spotted onto a MALDI target with an equal volume of cyano-4-hydroxycinnamic acid (10 mg/ml; Sigma) saturated with 50%acetonitrile in 0.05% TFA. The mass spectrometric (MS) spectra were acquired on a Voyager DE STR MALDI-TOF mass spectrometer (ABI, Foster City, CA). The instrument settings were reflector mode with 160-ns delay extraction time, positive polarity, and 20-kV accelerating voltage. The spectrum mass-tocharge (m/z) were usually acquired with laser shots at 200/spectrum and ranged from 1000 to 4000 m/z. External calibration was performed using a Peptide Mass Standard kit (Perspective Biosystems, Framingham, MA). The acquired MS spectra were processed with base-line correction, noise removal (5%), and peak deisotoping using DataExplore (Version 4.0.0.0) software to generate the peaklist for peptide mass fingerprint (PMF) analysis. The PMF data were input into Mascot (http://www.matrixscience.com/) for protein identity searching. The PMF data from the Voyager DE STR mass spectrometer were searched against the NCBInr database (release date January 7, 2012; 16,831,682 sequences, 5,781,564,572 residues; Homo sapiens 241,934 sequences). The search was restricted to “Human” as taxonomy. The other search parameters included type of search (peptide mass fingerprint), enzyme (trypsin), fixed modifications (carbamidomethylcysteine), variable modifications (methionine oxidation), mass values (monoisotopic), protein mass (unrestricted), peptide mass tolerance (100 ppm), peptide charge state (1+), and 1 maximum missed cleavages. Proteins whose scores were greater than 66 were considered as significance (p < 0.05), and only human proteins with the best score in each Mascot search were accepted as successful identifications. The coverage of amino acid sequence, the Mascot score, the mass and pI, and accession number are obtained for each protein.
Proteins in a 2D gel spot that was not identified or was a mixture identified by MALDI-TOF PMF data were subjected to electrospray ionization (ESI)-quadrople (Q)-TOF MS/MS analysis. Briefly, the tryptic peptides from 2D gel spots were loaded onto a C18 pre-column for concentrations and fast desalting, and then eluted to the reversed-phase column for separation. MS/MS spectra were performed in data-depended mode in which up to four precursor ions above an intensity threshold of 7 counts/seconds (cps) were selected for MS/MS analysis from each survey scan. For MS/MS database query, the peptide sequence tag (PKL) format file that was generated from MS/MS data with MassLynx v 4.0 software were inputted into the Mascot search engine to search protein against the NCBInr database (release date January 7, 2012; 16,831,682 sequences, 5,781,564,572 residues; Homo sapiens 241,934 sequences). A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P < 0.05). The false-positive rate was calculated with 2∗ reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 45 and a false-positive rate was approximately 3.1%. Only protein IDH3B was identified with only one peptide with a Mascot score of 73, its MS/MS spectrum was further checked manually and interpreted with de-novo sequencing.
Functional enrichment analyses of mitochondrial DEPs and protein-protein interaction data
The gene names of mitochondrial DEPs were converted to NCBI-Entrez format for consistency and saved as a text file that was inputted into Cytoscape v 2.8.2 (http://www.cytoscape.org), and BiNGO plugin 2.44 downloaded from Cytoscape manage plugin was used to analyze the enriched biological processes and molecular functions. Human protein-protein interaction data that were downloaded from HPRD (release 9; http://www.hprd.org) and NCBI databases (http://www.ncbi.nlm.nih.gov) were used to generate a protein-protein interaction sub-network of mitochondrial DEPs; the result only extended one layer connection and could be visualized by Cytoscape.
siRNA transient transfection
The siRNA transient transfections of metastatic 5-8F cells were conducted according to siRNA Transfection Protocols (Santa Cruz) and Transfection Technology Protocols (Invitrogen) with a minor modification. Briefly, the cells were incubated in a six wells plate (Costar) at 37°C in a CO2 incubator till 70-80% confluence. The solution A (2.4 μl RNAiMAX in 100 μl serum-free medium) and solution B (10 μl siRNAs duplex in 100 μl serum-free medium) were mixed gently (the final concentration in each well was 100 nM), and incubated (room temperature; 45 min). Cells were washed twice with serum-free medium, and then added into 200 μl of above A + B mixture and 800 μl of serum-free medium in each well. After cells were incubated (7 h), the medium in each well were removed and replaced with 2.5 ml normal fresh growth medium, cells were continued to be incubated (48 h). Cells were washed twice with D-Hank’s solution, digested with 0.25% trypsin and 0.02% EDTA, collected by centrifugation (400 × g; 10 min; 4°C). A scrambled sequence control siRNA experiment was conducted at the same time. The efficiency of siRNA transfection was tested by Western blotting.
Transwell Migration assays
Cells Transwell Migration assay was carried out according to Matrigel Basement Membrane Matrix Product Specification Sheet (BD Biosciences) and Transwell Insert Product Description (Corning) with a minor modification. Briefly, the Matrigel basement membrane matrix was thawed (4°C; overnight), the Transwell plates was put and inserted (both were added with serum-free medium) in a CO2 incubator (37°C; 2 h). The medium in Transwell plates was removed and Transwell plates were inserted and kept on ice. Pre-cooled pipettes were used to add 20 μl Matrigel matrix (1:1 diluted with pre-cooled serum-free medium) into each compartment of Transwell inserts and swirled immediately to disperse materials evenly, placed Transwell inserts (37°C; 30 min) to form thin Matrigel. A volume (600 μl) of medium (RPMI 1640 with 10% FBS and 5 μg/ml fibronectin) was added to each Transwell plates well, followed by adding the above thin Matrigel inserts, then 200 μl medium and the cells (resuspended cells in medium at a density of 5 × 104 cells/ml) were added into each compartment of thin Matrigel inserts, and incubated in a CO2 incubator (37°C; 24 h). All of these experiments should be carefully handled using sterile techniques. Those non-migrated cells on each compartment of thin Matrigel inserts were scraped off with cotton swabs, the migrated cells were fixed and stained by 0.1% crystal violet solution. The cells without siRNA transient transfection and with a scrambled sequence transfection were used as control groups. In three independent experiments, the number of cells that migrated through Transwell insert membrane was counted in 9 visual fields per well at 400 × under an Olympus microscope, means of these counts were calculated for statistical analysis using SPSS software, and variables were compared using Student’s t-test with a significance level of P = 0.05.
