Nucleolin Participates in DNA Damage Response Figure 4. Nucleolus-related function of nucleolin might be dispensable for of its role in the DNA damage response. (A and B) U2OS cells were transfected by nucleophosmin (B23) siRNA, and after 2 days these cells were irradiated by 5 Gy of c-ray. After 30 minutes, their cells were fixed for immuno-staining (A), while their cells were harvested at indicated time for Western blot analysis (B). (C) U2OS cells were irradiated by 5 Gy of c-ray with or without pre-treatment of actinomycin D (0.02 mg/ml, 1 hour). These cells were harvested at indicated times after IR and analyzed by Western blot using indicated antibodies. doi:10.1371/journal.pone.0049245.g004 show chromatin association without DNA damage (Fig. 6A). However, nucleolin-depleted cells did not accumulate Rad51 and RPA with or without DNA damage. ATM and the NHEJ factor, KU80 also showed a similar deficiency (Fig. S6A). They accumulated into chromatin fraction in control cells after irradiation, but depletion of nucleolin abrogated these accumula- tions, in agreement with the deficiency of DNA-PK auto- phosphorylation and ATM-dependent phosphorylation in knock- down cells (Fig. 3C and S4A). Recent reports have shown that ubiquitination of histone H2A/H2AX is important for DSB damage response, particularly HR repair pathway [19]. Hence, we examined H2A ubiquitination-related pathway in nucleolin-de- pleted cells. Nucleolin knockdown decreased both ubiquitination of H2A and c-H2AX and focus formation of RNF168 E3 ligase (Fig. 6B and S6C), which is responsible for H2A/H2AX poly- ubiquitination. MDC1 is important for accumulation of RNF8/ RNF168 to DNA damage sites, but IR-induced chromatin association of MDC1 was also reduced (Fig. S6C). Furthermore, PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49245