Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 with either pZeo HA HuR or pZeo empty vector using Lipofectamine 2000 (Invitrogen). After five days trans- fected media was removed and replaced with fresh med- ium containing 200 μg/ml of Zeocin antibiotic (Invitrogen). Cells were selected for a ten day period. After ten days, the selected cells were maintained in 50 μg/ml of Zeocin to maintain pZeo HA HuR and empty vector expression. No viable cells remained in the untransfected well. Cells were then cloned by limiting dilution. Lentiviral RNAi HuR knock-down In order to knockdown HuR, PSICOOLIGOMAKER v1.5 http://web.mit.edu/ccr/labs/jacks/was used to identify optimal shRNAs sequences to HuR. We tested multiple sequences and chose GGATCCTCTGGCA- GATGT, identified and designated shRNA H760. Annealed sense and antisense DNA (Integrated DNA Technologies, Inc, IDT, Coralville, IA), along with stem loops to create hairpin, were cloned into the HpaI and XhoI restriction sites in the Lentilox pll3.7 vector (ATCC). After sequence verification, lentivirus was packaged in 293FT cells using ViraPower™ Lentiviral Expression Systems (Invitrogen) following manufac- turer’s protocol. Both MB-231 and MCF-7 cells were seeded at a density of 100,000 cells in 100 mm tissue culture plates with 10 ml of media. The following day lentivirus, expressing either GFP and no shRNA (empty lentilox control) or GFP and HuR shRNA H760, was added at a multiplicity of infection (MOI) of 10 along with polybrene (8 μg/ml) (Sigma-Aldrich Corp, St. Louis, MO). After five days, cells were harvested by trypsinization and sorted for GFP expression using BD FACSDiva (BD Bioscience). Cells were cloned by limit- ing dilution and GFP expression was assessed using FACScan (BD Bioscience) and CellQuest software (BD Bioscience). GFP expression was >98% and indicated homogenous cell population. SDS-PAGE and Western Blot Analysis Western analysis was performed as described previously with slight modifications [47]. Briefly, cells were har- vested and lysed in triple-detergent RIPA buffer with protease inhibitor cocktail (Roche, Pleasanton, CA). For nuclear and cytoplasmic fractionation, the NE-PER kit was used (Pierce, Rockford, IL). Protein quantity was determined by Bradford Assay. Forty μg of protein was electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The mem- brane was blocked with 5% nonfat milk at room tem- perature for 1 hr and incubated with anti-b-tubulin (1 μg/ml, Sigma-Aldrich) at 4°C overnight. After washing, the membrane was incubated with monoclonal anti- HuR clone 3A2 antibody (1 μg/ml) at room temperature Page 4 of 15 for 1 hr, or anti-CD9 antibody (1:100) (Santa Cruz Bio- technology, Inc., Santa Cruz, CA) at 4°C overnight. The secondary antibody used was sheep anti-mouse Ig horse radish peroxidase (1:4000) (GE Healthcare, Piscataway, NJ), incubated at room temperature for 1 hr. Specific proteins were detected using chemiluminescence (GE Healthcare). HuR knock-down was determined to be >90% using Bio-Rad’s Quantity One software (Bio-Rad) normalizing to b-tubulin, and HuR over-expression was quantitated in a similar manner. Biotin Pull-down Biotinylated transcripts were synthesized using cDNA that was prepared from MB-231 cells. Templates were prepared using forward primers that contained the T7 RNA polymerase promoter sequence (CCAAGCTTC- TAATACGACTCACTATAGGGAGA [T7]). Primers used for the preparation of biotinylated transcripts span- ning the CD9 CR, and 3’UTR (NM_001769) and CALM2 CR and 3’UTR (NM_001743.3) were as follows: CD9 CR 118-560: [T7] TCAAAGGAGGCAC- CAAGTGCAT and AACGCATAGTGGATGGCTTTCA CD93’UTR798-1231: [T7] AGTCAGCTTACATC- CCTGAGCA and GACATTGTCATAATTTTTTAT- TATGTATC CALM2 CR 72-515: [T7] GCTGACCAACTGACT- GAAGA and CTTTGCTGTCATCATTTGTACAAA CALM2 3’UTR 518-1128: [T7] AGACCTTGTACA- GAATGTGTTAA and GGGTAAATTGTAATTTTT- TTATTGGAA GAPDH 3’UTR: [T7] CCTCAACGACCACTTTGTCA and GGTTGAGCACAGGG TACTTTATT The PCR-amplified fragments were purified and used as templates for in vitro synthesis of the corresponding biotinylated RNAs by MAXIscript kit (Ambion®, Applied Biosystems). Biotin pull-down assays were performed by incubating 40 μg of MB-231 cell lysates with equimolar of biotinylated transcripts for 1 hr at room temperature. The complexes were isolated using paramagnetic strep- tavidin-conjugated Dynabeads (Dynal®, Invitrogen), and bound proteins in the pull-down material were analyzed by Western blotting using an antibody recognizing HuR (Santa Cruz). After secondary-antibody incubations, the signals were visualized by chemiluminescence (Amer- sham Biosciences, GE Healthcare). Statistical Analysis of Microarray Data Analysis of microarray gene expression data was primar- ily performed using the Linear Models for Microarray Data (limma) package [49] and the lumi package [50], available through the Bioconductor project [51] for use with R statistical software [52]. After data pre-processing was completed (Appendix), the statistical analysis was performed using moderated t-statistics applied to the