As described above, nucleolin is one of the most abundant non- ribosomal proteins of the nucleolus and is important for nucleolar formation and rRNA metabolism. Hence, we investigated the possibility that the defect in DNA damage responses for depletion by siRNA is due to the deficiency of normal nucleoli formation in those cells. However, both knockdown of nucleophosmin, a binding partner for nucleolin, and repression of rRNA synthesis by actinomycin D did not show any defect in the DNA damage response (Fig. 4). These results suggest that the function of nucleolin in the DNA damage response is distinct from its role in the nucleolus. It is known that several DNA repair-related proteins such as WRN, BLM, RECQL4, and aprataxin mainly localizes in the nucleolus and many DNA repair proteins are present in the nucleolus fraction [44]. Nucleolus contains high copies of ribosomal RNA gene (rDNA) and the maintenance of this copy number is important for genome integrity in yeast [45,46]. Moreover, amplification of rDNA requires HR-related proteins such as RAD52 and MRE11 [47]. These reports suggest the importance of HR pathway for rDNA maintenance and the existence of nucleolus-specific DNA repair mechanisms. In fact, it was reported in yeast that the Smc5-Smc6 complex and sumoylated Rad52 regulate recombination repair at the ribosomal gene locus, and that the maintenance of rDNA could be performed by perinuclear chromosome tethering system [47]. Hence, mammalian cells may also have a specific system for rDNA maintenance using HR. As our results show here that nucleolin could participate in HR repair for genome integrity, nucleolin may also contribute to the repair system for rDNA. Several observa- tions suggest that nucleolin is up-regulated in human tumors and overexpression of nucleolin cooperates with oncogenic mutated Ras in transformation of rat embryonic fibroblast and human cancers [28,48]. Therefore, it will be important to further clarify the role of nucleolin in genome integrity and rDNA maintenance. Materials and Methods Cell Culture HeLa, U2OS, hTERT-immortalized human fibroblast 48BR [49], were cultured in DMEM (Sigma) supplemented with 10% FBS (Invitrogen) and antibiotics. H2ax 2/2 and H2ax +/2 mouse fibroblasts were supplied by Dr. A. Nussenzweig and were cultured under above condition. Normal human lymphoblastoid GM2184 [21] were cultured in RPMI (Sigma) supplemented with 10% FBS (Invitrogen) and antibiotics. Antibodies Phospho-ATM (S1981) mouse monoclonal and c-H2AX mouse monoclonal antibodies (Millipore Co.), phospho-SMC1 (S966) rabbit polyclonal and SMC1 rabbit polyclonal, MDC1 rabbit polyclonal and Phospho RPA32 (S4/S8) rabbit polyclonal antibodies (Bethyl Laboratories Inc.), phospho-p53 (S15) mouse monoclonal, phospho-Chk2 (T68) rabbit polyclonal antibodies (Cell Signaling Technology), hMre11 rabbit polyclonal and Nbs1 rabbit polyclonal antibodies (Novus Biologicals), and nucleolin rabbit polyclonal and mouse monoclonal and p53 mouse mono- clonal antibodies (Santa Cruz Biotechnology), and 53BP1 rabbit polyclonal, BRCA1 mouse monoclonal and Rad51 rabbit poly- clonal antibodies (Merk Co.) and anti-RPA antibody (for RPA34; Calbiochem) and anti-Ubiquitil-Histone H2A (Upstate) were used for Western blot analysis or immuno-staining. Anti-RNF168 antibody was supplied by Dr. D. Durocher. Nucleolin Participates in DNA Damage Response SiRNA Knockdown Experiments Sub-confluent cells, seeded culture dishes the day before, were transfected with nucleolin siRNA (Be-Bridge International Inc. and QIAGEN Co.) nucleophosmin siRNA (Santa Cruz Co.), or negative control siRNA (Be-Bridge International Inc.) using lipofectamine 2000 (Invitrogen Life Technology). After 2 days, these cells were re-seeded in proper culture dishes. Next day, these cells were used for Western blot or immunofluorescence, HR and NHEJ analysis. GST-pull-down and Mass Spectrometry Analysis GST-fused human H2AX (wild type) was generated by insertion of PCR product of human H2AX cDNA into pGEX-2T (GE healthcare CO). GST-fused mutated H2AX (S139E) was gener- ated by PCR using primers containing mutations within the codon for serine 139. Purified GST-fused proteins were used for pull- down of interacting proteins from HeLa nuclear extracts as previously reported [21]. These candidate proteins were identified using general peptide mass fingerprint analysis by autoFLEX (Bruker Dartonics). Laser Micro-irradiation Cells were transfected with EGFP-nucleolin plasmid, supplied by Dr. J.A. Borowiec (26). After two days, laser micro-irradiation analysis was performed as previously described (24) using a con- focal laser microscopy (TCS-SP5; Leica Co.). Chromatin Immunoprecipitation (ChIP) Assay To generate DSBs by I-SceI restriction enzyme, 16106 HeLa- DRGFP cells were pre-treated with 10 mM of Nu7026 (DNA- PKcs inhibitor; Calbiochem) for 1 hour and then, 50 mg of the I- SceI expression vector pCBASce was introduced to them by electroporation (BIO-RAD). At 1 day after transfection, cells on dishes were treated with 1% formaldehyde at 37 C for 10 min followed by the addition of 0.125 M glycine, after that their cells were harvested by a scraper. Chromatin Immunoprecipitation assay using their harvested cells was performed previously reported [38]. The precipitated DNA were then dissolved in 50 ml of TE and analyzed by quantitative real-time PCR with the ABI PRISM7500 system using Power SYBR Green PCR Master Mix (Applied Biosystems). Primers used for detection of the I-SceI break site were Sce180-F (59-CATGCCCGAAGGCTACGT) and Sce180-R (59-CGGCGCGGGTCTTGTA). As an internal con- trol for normalization of the specific fragments amplified, human GAPDH locus was amplified using whole genomic DNA with GAPDH-F (59-TCTCCCCACACACATGCACTT) and GAPDH-R (59-CCTAGTCCCAGGGCTTTGATT). HR and NHEJ Analysis HR and NHEJ analysis was performed as previously reported [7]. To measure the HR or NHEJ repair of I-SceI-generated DSBs, 50 mg of the I-SceI expression vector (pCBASce) was introduced to 1000000 Hela-DRGFP (for HR), NBS-DRGFP (for HR) or MRC5SV-pEJ (for NHEJ) cells cells, by electroporation (GenePulser; BIO-RAD). To determine the amount of HR or NHEJ repair, the percentage of GFP-positive cells was quantified by flow cytometer at 3 days after electroporation with FACAca- libur (Becton Dickinson). Preparation of Chromatin Fraction The cell lysates with or without DNA damaging treatment were fractionated into cytoplasmic solution and nuclei. Separated nuclei was resuspended into 2 6 RIPA buffer (DNA damage-treated or PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49245