Human breast cancer cell lines
The human breast cancer cell lines SK-BR-3, AU565, HCC1569, HCC70, and MCF7 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The Herceptin-resistant human breast cancer cell line JIMT-1 was purchased from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). SK-BR-3 HR cells, which are resistant to Herceptin (Genentech, Inc., South San Francisco, CA), were spontaneously derived from SK-BR-3 cells by increasing the generation-to-generation passage.
Genotyping of SK-BR-3 and its Herceptin-resistant-derivative cell line, SK-BR-3 HR
The total amount of genomic DNA was extracted using the TRI reagent according to the manufacturer's instructions (Molecular Research Center, Cincinnati, OH, USA). PCR amplification was performed using the AmpFlSTR Profiler™ PCR Amplification Kit (Applied Biosystems, Oster City, CA) to amplify the D3S1358, vWA, FGA, amelogenin, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820 loci in sample genomic DNA. PCR assays were performed according to the manufacturer's recommendations except that the PCR volume was 50 μL and contained 2 ng of template DNA. The PCR conditions were 95°C for 11 minutes, followed by 28 cycles at 94°C for 1 minute, 59°C for 1 minute, and 72°C for 1 minute. A final extension was conducted at 60°C for 45 minutes, and the products were then maintained at 25°C. Separation and detection of the amplified product was performed using an ABI 3730 Genetic Analyser (Applied Biosystems) with GeneScan 2.1 and Genotyper 2.0 software.
3- [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
A colorimetric assay using the tetrazolium salt MTT was used to assess cell proliferation after HSP27 suppression. MTT assays were performed as described in a previous report . Briefly, 0.18 mL cultures containing equal numbers of cells were incubated in each well, to which 0.02 mL of 10 × Herceptin (Genentech, Inc.) or phosphate-buffered saline (PBS, an untreated 100% survival control) was added. After 4 days of culture, 0.1 mg of MTT was added to each well, and incubated at 37°C for a further 4 hours. Plates were centrifuged at 450 × g for 5 minutes at room temperature and the supernatant was removed. Dimethyl sulfoxide (0.15 mL) was added to each well to solubilize the crystals, and the plates were immediately read at 540 nm using a scanning multiwell spectrometer (Bio-Tek instruments Inc., Burlington, VT). All experiments were performed three times, and the IC50 (μg/mL) values are presented as means ± standard deviation.
Western-blot analysis was performed as described previously . Briefly, cell homogenates containing equivalent amounts of protein were centrifuged at 4,000 × g, and the supernatant fractions were separated by SDS-PAGE. Following electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA) blocked by incubation for 2 hours at 4°C in 1% Tween 20-TBS buffer containing 1.5% non-fat dry milk (Bio-Rad, Richmond, USA) and 1 mM of MgCl2. Membranes were incubated for 2 hours at room temperature with primary antibodies against Her2 (Dako, Glostrup, Denmark), HSP27 (Abcam, Cambridgeshire, UK), fibrillarin (Abcam), or actin (Sigma, St Louis, MO). Membranes were washed three times with blocking solution, for 15 minutes at a time, and incubated with diluted horseradish-peroxidase (HRP)-conjugated secondary antibody (Southern Biotech, Birmingham, AL) for 1 hour at room temperature. This was followed by washing with blocking solution (3 × 15 minutes), incubation with WEST-ZOL® plus chemiluminescence reagent (iNtRON Biotechnology, Gyeonggi, Korea) for 1 minute, and exposure to film (Kodak Blue XB-1, Rochester, NY).
Two-dimensional electrophoresis (2-DE) analysis was performed as previously described . Briefly, 0.15 mg protein samples were applied to 13 cm immobilized pH 3–10 non-linear gradient strips (Pharmacia Biotechnology, Uppsala, Sweden). Proteins were focused at 8,000 V within 3 hours. The second-dimension separation involved 12% polyacrylamide gels (chemicals from Serva, Heidelberg, Germany and Bio-Rad, Hercules, CA). 2-DE gels were stained with Colloidal Coomassie Blue (Invitrogen, Carlsbad, CA) for 24 hours and were then destained with deionized water.
Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and database searching
Sections of 2-DE gels containing proteins of interest were excised, destained with 50% acetonitrile in 0.1 M ammonium bicarbonate, and dried in a SpeedVac evaporator. Dried gel pieces were reswollen with 30 μL of 25 mM sodium bicarbonate at pH 8.8, containing 50 ng of trypsin (Promega, Madison, WI) at 37°C overnight. α-Cyano 4 hydroxycinnamic acid (20 mg) (Bruker Daltonics, Bremen, Germany) was dissolved in 1 mL acetone:ethanol (1:2, v/v), and 0.5 μL of the matrix solution was mixed with an equivalent volume of the sample. Analysis was performed using an Ultraflex TOF/TOF system (Bruker Daltonics).
The Ultraflex TOF/TOF system was operated in the positive-ion-reflect mode. Each spectrum was the average of 250–450 laser shots. Mass spectra were first calibrated in the closed external mode using the peptide calibration standard II (Bruker Daltonics), and sometimes also in the internal statistical mode to achieve maximum calibration mass accuracy, and then analyzed with FlexAnalysis software, version 2.4 (Bruker Daltonics). Peptide mass peaks from each spectrum were analyzed by the Mascot peptide mass fingerprinting search tool http://www.matrixscience.com using BioTools software, version 3.0 (Bruker Daltonics).
The search included peaks with a signal-to-noise (S/N) ratio greater than 3. The peak list for each sample was compared with the non-redundant Mass Spectrometry Protein Sequence Database (MSDB) for protein identification. Standard settings included the following: enzyme, trypsin; missed cleavage, one; fixed modifications, none selected; variable modifications, oxidized methionine; protein mass, blank; mass values, MH+ (mono-isotopic); mass tolerance, between 75 and 100 ppm.
Nuclear fractions of cells were isolated from cytoplasm using the nuclei isolation kit 'Nuclei PURE Prep' (Sigma-Aldrich, St Louis, MO), as recommended by the manufacturer. Membrane fractionation was performed as described previously with the following modifications . Briefly, cells were vortexed vigorously for 5 min in cold lysis buffer containing 40 mM of Tris and 150 U/mL of benzonase, and then homogenized on ice. Vortexing and homogenization were repeated. The cell homogenate was centrifuged at 12,000 × g for 10 min at room temperature. The insoluble pellet was washed twice with lysis buffer and resuspended in membrane fractionation buffer containing 5 M urea, 2 M thiourea, 2% w/v CHAPS, 40 mM Tris and 100 mM DTT. The vortexing, homogenization and centrifugation process was then repeated. The supernatant was collected as the membrane fraction.
All procedures were performed at 4°C unless otherwise specified. Approximately 107 cells in 1 ml of cold 1 × RIPA buffer containing protease inhibitors (Roche Diagnostics) were incubated on ice for 30 minutes with occasional mixing. Cell lysates were centrifuged at 12,000 × g for 10 minutes, and the supernatants were collected without disturbing the pellet. The supernatants were mixed with primary antibodies against Her2 (Dako) and HSP27 (Abcam), and incubated for 2 hours on a rocking platform. Prepared protein G sepharose beads (100 μL) (GE Health Care Life Sciences) were added and were further incubated on ice for 1 hour on a rocking platform. The mixture was centrifuged at 10,000 × g for 30 seconds, and the supernatant was then completely removed. Protein G sepharose beads were washed five times with 1 mL of cold 1 × RIPA to minimize the background. Next, 100 μL of 2 × SDS sample buffer was added to the bead pellet, and the mixture was heated at 100°C for 10 minutes. After boiling, immunoprecipitates were centrifuged at 10,000 × g for 5 minutes, and the supernatants were collected for Western-blot analyses.
siRNA synthesis and transfection
Validated siRNA (Cat No. SI00300496) against HSP27 and non-silencing (NS) control siRNA (5'-AATTCTCCGAACGTGTCACGT-3') were obtained from Qiagen, Chatsworth, CA. Transfection with siRNAs was performed using HiferFect transfection reagent (Qiagen), according to the manufacturer's instructions. Briefly, 2 μL of 20 μM siRNA solution and 20 mL of the transfection reagent were incubated in 100 mL of serum-free RPMI 1,640 medium for 10 minutes to facilitate complex formation. The resulting mixture (final concentration of 5 nM) was added to the Herceptin-resistant SK-BR-3 (4 × 105), and incubated in a 60 mm tissue culture dish with 4 mL RPMI 1640.