metaphase [44], initiation of centrosome separation at G2/M [33], and centriole disengagement late in mitosis [45,46]. Duensing and colleagues demonstrated that treatment of U2OS cells with Z- L3VS, which inhibits the trypsin, chymotrypsin, and peptidylglu- tamyl peptidase activities of the proteasome, led to centrosome over-duplication characterized by a mother centriole surrounded by a rosette of daughter centrioles [1]. These data illustrate that a functional proteasome is essential for centrosome homeostasis. In light of our observations demonstrating that centrin accumulates in the presence of proteasome inhibitors, we speculate that lack of centrin degradation could contribute to the proteasome inhibitor- induced centrosome aberrations. Interestingly while we were performing revisions on this manuscript, Yang et al. demonstrated that Mps1 phosphorylates centrin-2 at three sites (T45, T47, and T118 or centrinTTT) in vitro [47]. The authors demonstrate that over-expressing GFP-tagged centrin-2 causes excess centrioles and that non-phosphorylatable centrin mutants, centrinAAT and centrinTTA, greatly reduce the over-production of centrioles [47]. Phospho-mimicking centrin mutants, centrinDDT, centrinTTD, and centrinDDD, showed increased formation of excess centrioles, but all three phosphor- ylation sites were required [47]. Previously the Fisk lab demonstrated that non-degradable centrosomal Mps1 can cause centriole over-production [48]; and in this manuscript, they demonstrate that centrin-2 is required for this process [47]. Interestingly by further studying the over-production of centrioles, Yang et al. demonstrated that over-expression phospho-mimicking centrins mutants can not compensate for a reduction in Mps1 in this process [47]. Because the triple centrin-2 phospho-mimic can’t compensate for lack of Mps1, this argues that there are other unidentified phosphorylation sites within centrin-2 or other Mps1 substrates that contribute to this process [47]. These along with other recent findings [49] suggest that centrin-2 regulation including its role in centrosome homeostasis could be far more complicated than originally appreciated. Materials and Methods Ethics Statement Frozen breast tissues were collected by the Mayo Clinic Breast SPORE Program under the Mayo Clinic Internal Review Board approval # 08-008233. This IRB-approved Protocol allows for use of tissues collected during standard patient care procedures without patient consent, therefore no consent was obtained. ‘‘Continuation of the above referenced study is approved by expedited review procedures (45 CFR 46.110, item 5). The Reviewer determined the research continues to pose no more than minimal risk to subjects. The Reviewer determined that this research continues to satisfy the requirements of 45 CFR 46.111.’’ Reagents All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Antibodies Antibodies were purchased from the following sources: monoclonal Aurora A clone 35C1 (AbD Serotec; Raleigh, NC), polyclonal Aurora A, goat polyclonal cdc16, monoclonal and polyclonal cdc20, and monoclonal cdc27 (Santa Cruz Biotechnol- ogy Inc.; Santa Cruz, CA), cyclin B (Cell Signaling Technology; Danvers, MA), HA-tag (Covance; Princeton, NJ), and b-actin (Sigma). Monoclonal centrin (20H5), polyclonal centrin (MC1), and p-S170 phospho-centrin antibodies were created in the Salisbury laboratory. Control of Centrin Stability by Aurora A Cell culture HeLa cells were maintained in DMEM with 10% FBS, 100 IU/ ml penicillin, and 100 mg/ml streptomycin. HeLa Tet-On cells were cultured in DMEM with 10% Tet System Approved FBS (Clontech; Mountain View, CA), 100 IU/ml penicillin, 100 mg/ml streptomycin, 400 mg/ml hygromycin, and 100 mg/ml geneticin. To induce expression of HeLa Tet-On cells containing centrin constructs, doxycycline was added to the medium at a final concentration of 1 mg/ml. Cell synchronization techniques For the double thymidine/nocodazole synchronization, HeLa cells were grown to approximately 80% confluency, treated with 2 mM thymidine for 12–16 hours, and released into medium containing 24 mM deoxycytidine for 9 hours. The second block was done as described above except the deoxycytidine release was only 8 hours. Cells were treated with 0.1 mg/ml nocodazole for six hours, harvested by mitotic shake-off, washed, and allowed to release in nocodazole-free medium. For the nocodazole synchro- nization, cells were treated with 0.5 mg/ml nocodazole for 12– 16 hours and harvested as previously described. Western blotting Cells were lysed in 1% SDS, Tris pH 7.4 lysis buffer supplemented with protease and phosphatase inhibitors and sonicated three times for five seconds/burst. Protein concentration was determined using the BioRad DC Assay. For whole cell lysate Western blotting, 20 mg of protein was separated on 10% or 15% SDS-PAGE gels. After separation, proteins were transferred to PVDF membrane using a semi-dry transfer apparatus (The W.E.P. Company, Seattle, WA), fixed with 0.2% glutaraldehyde for 45 minutes at room temperature, blocked with 5% non-fat dry milk for at least 1 hour at room temperature, and incubated with the indicated antibodies overnight at 4uC. Membranes were incubated in secondary antibodies (GE Healthcare; United Kingdom) diluted to 1:2500 for one hour at room temperature and visualized with ECL (ECL anti-rabbit or anti-mouse IgG- HRP; GE Healthcare; anti-goat IgG-HRP; Santa Cruz) according to manufacturer’s instruction. Flow Cytometry Cells were harvested by trypsinization, fixed in 95% ethanol, incubated on ice for at least one hour, washed three times, and stained with propidium iodide (100 mg/ml in 0.1% sodium citrate). DNA content of cells was determined by measuring the propidium iodide staining on channel FL2 using a Becton Dickinson FACScan (San Jose, CA). Immunoprecipitations (IP) HeLa cells were grown to 85–90% confluence, lysed in 200 ml of mild lysis buffer (1% NP-40, 10 mM EDTA, 10 mM Tris-HCl, pH 7.4 with protease inhibitors and phosphatase inhibitors), scraped and collected into a microfuge tube. Cell lysates were sonicated three times at 5 seconds/bursts. IP buffer A (190 mM NaCl, 50 mM Tris-HCl, 6 mM EDTA, 2.5% Triton X-100, pH 7.4) was added to each tube to bring the cell lysate volume up to 1 ml. Each IP was spun down at 14,000 rpm for 5 minutes, and the supernatant was removed and placed into a new microfuge tube. Six ml of antibody was added to each IP and rocked overnight at 4uC. The next day, 50 ml of Pierce A/G beads (Pierce, Rockford, IL) was added to each IP and allowed to rock at 4uC for 1 hour. IPs were spun down at14,000 rpm for 1 minute to pellet the beads and washed 3 times with IP buffer A and 3 times PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21291