S2B). Introduction of H2AX-WT to H2AX (2/2) MEFs restored the recruitment of nucleolin to irradiated area, but mutated H2AX (S139A) did not (Fig. S2C). Thus, H2AX and its phosphorylation are important for the recruitment of nucleolin to DSBs. Recently, Chromatin-immunoprecipitation (ChIP) has been extensively and effectively used to investigate the recruitment and accumulation of DDR factors to DSB sites [32]. ChIP analysis revealed that nucleolin as well as hMRE11 and c-H2AX accumulate around DSB sites in both HeLa and MRC5SV cells (Fig. 2C and S2E). Take together, these results indicate that nucleolin can be recruited to DNA damage sites and may function in the DNA damage response. Nucleolin is Required for ATM-dependent Cell Cycle Checkpoint Implementation In order to clarify the role of nucleolin in DDR, we designed siRNA targeting human nucleolin, which effectively reduced the expression of nucleolin protein (Fig. S3A). Although we identified nucleolin as an H2AX-interacting protein, U2OS cells with knockdown of nucleolin showed normal c-H2AX foci formation after irradiation (Fig. 3A and S3B). MDC1, NBS1 and MRE11 also showed normal focus formation following irradiation. Very interestingly, focus formation by phosphorylated-ATM was re- duced in nucleolin-knockdown U2OS cells (Fig. 3AB and S3C), and similar results were observed in HeLa cells (Fig. S3D). As auto-phosphorylation of ATM is known to regulate its kinase activity and subsequent cell cycle checkpoints, we interrogated the nucleolin-depleted cells for defects in ATM-mediated DNA damage responses. Nucleolin-depleted 48BR and MRC5SV cells exhibited reduced ATM auto-phosphorylation and reduced phosphorylation of its substrates SMC1, NBS1, p53, Chk2, RPA34, DNA-PKcs and c-H2AX, but depletion of nucleolin did not influence the expression of their proteins (Fig. 3C and S4AB). A different nucleolin siRNA (nucleolin siRNA2) also remarkably reduced nucleolin and repressed the phosphorylation of ATM and SMC1 (Fig. S4C), suggesting that the results in Figures 3C and S4A are not due to off-targets effects of the siRNA being used. Furthermore, nucleolin-knockdown abrogated G2 checkpoint after irradiation, although accumulation in G2 phase was observed 12 hours after irradiation of control cells with 10 Gy of IR (Fig. S4D). Furthermore, formation of 53BP1 foci following IR was also repressed in nucleolin-knockdown cells (Fig. 3AB and S3CD), although ATM-deficient AT cells can still form 53BP1 foci in response to IR [33]. Probably, nucleolin could participate in ATM-dependent DSB damage responses and independent-DSB responses such as 53BP1 focus formation. Deficiency in DNA Damage Response Upon Nucleolin Knockdown is not Due to its Nucleolar Function Nucleolin forms a complex with nucleophosmin and this complex functions in nucleolus formation and ribosomal RNA (rRNA) synthesis [25]. Besides, recent reports suggest that nucleophosmin (B23) is important for translocation of p19ARF in response to DNA damage [34]. Hence, we investigated whether repression of nucleophosmin by siRNA causes effects similar to nucleolin knockdown. siRNA-mediated depletion of nucleophos- min did not affect the formation of 53BP1 foci after irradiation (Fig. 4A), contrary to the abrogation of 53BP1 foci seen after nucleolin knockdown (Fig. 3AB). ATM-dependent phosphoryla- tion was also not influenced by nucleophosmin knockdown (Fig. 4B), suggesting that the defective DNA damage responses in nucleolin-depleted cells are independent of its binding partner, nucleophosmin. As nucleolin is also important for rRNA synthesis, Nucleolin Participates in DNA Damage Response we also evaluated whether a deficiency in rRNA synthesis might abrogate ATM-related DNA damage responses (Fig. 4C). Treat- ment of mammalian cells with low concentrations of actinomycin D (0.02 mg/ml) repressed rRNA synthesis [35], but this treatment did not change the phosphorylations of ATM, SMC1, p53 and H2AX (Fig. 4C). Taken together, nucleolin could play a role in the DNA damage response due to a specific novel function, which is distinct from its role in nucleolus formation and rRNA synthesis. Nucleolin Could Participate in DNA Double-strand Break Repair DNA double-strand break repair is very important for cell survival after the induction of DSBs. DSB repair involves two major pathways, NHEJ and HR. In order to elucidate the role of nucleolin in DSB repair, we first examined the interaction of nucleolin with DSB repair factors. Fig. 5A shows that anti- nucleolin antibody co-precipitated important HR factors, RPA34 and NBS1, suggesting a role of nucleolin in the HR pathway. We also estimated the importance of nucleolin in HR activity using a GFP-based HR reporter system [36] after siRNA-mediated knockdown of nucleolin (Fig. 5B). The generation of DSB by expression of I-SceI restriction enzyme induced approximate 8% of GFP-positive cells via HR repair pathway, but nucleolin repression by siRNA decreased this activity by about half. As NBS1 is important factor for HR repair, NBS1-deficient NBS cells also show a low frequency of GFP-positive cells (approximate 2%; Fig. 5B). Repression of nucleolin in NBS cells did not further decrease this HR frequency, suggesting that nucleolin could function for HR repair in an NBS1-related pathway. Importantly, IR-induced focus formation of HR factors such as Rad51 and BRCA1 was also reduced in nucleolin-depleted cells (Fig. 5C and S5A). Resection of DSB ends is one of critical initial reaction for HR repair pathway, and this resection reaction could be visualized by RPA34 focus. When we observed the focus formation of RPA34 following irradiation, formation of RPA34 focus was repressed in nucleolin-depleted cells (Figure 5D and S5C). Reduction of RPA34 phosphorylation in nucleolin-depleted cells (Fig. 3C) also supports the role of nucleolin in HR repair. Although nucleolin is also important for transcription and translation, the repression of nucleolin did not change the expressions of HR factors (Fig. S5B), indicating that nucleolin could participate in HR machinery directly, perhaps by facilitating the initial resection and subsequent recruitment/accumulation of key HR proteins such as RPA, Rad51 and BRCA1 at DSB sites. As nucleolin interacts with KU70 (Fig. 1C), we also examined NHEJ activity using a GFP-based NHEJ reporter system [37]. Fig. 5B shows that the generation of DSB by I-SceI induced approximately 6% of GFP-positive cells via NHEJ repair pathway, but the repression of nucleolin reduced this activity, suggesting that nucleolin might also contribute to NHEJ repair partially. As these results suggest the contribution of nucleolin to both HR and NHEJ repair pathways, we estimated the deficiency of DSB repair in nucleolin-deficient cells using c-H2AX foci as a surrogate marker for DSBs (Fig. 5E and S5D). At 15 minutes after exposure to 2 Gy of c-ray, more than 90% of both control and the deficient cells showed c-H2AX foci, but after 6 hours, most control cells exhibited diminished c-H2AX foci. However, nucleolin-deficient cells retained c-H2AX foci after 6 hours, indicating attenuated DSB repair. Similar results were obtained when NBS1 foci were used to quantify DSBs (Fig. S5E). Taken together, these results indicate that nucleolin is important for DNA double-strand break repair. PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49245