Nucleolin Participates in DNA Damage Response Figure 3. Nucleolin contributes to ATM-dependent DNA damage responses. (A and B) U2OS cells were transfected by nucleolin siRNA or negative control siRNA, and after 2 days these cells were irradiated by 5 Gy of c-ray. After 30 minutes, their cells were fixed and immuno-staining was performed using indicated antibodies. Percentage of phospho-ATM or 53BP1 foci-positive cell at indicated times after 2 Gy of irradiation was shown in (B). Open column: control, closed column: nucleolin siRNA. (C) 48BR cells were transfected by nucelolin siRNA. After 2 days, these cells were irradiated by 5 Gy of c-ray and were harvested at indicated times after IR and analyzed by Western blot using indicated antibodies. doi:10.1371/journal.pone.0049245.g003 Nucleolin is Required for MDC1-dependent Chromatin Modification and Histone Eviction As most of DDR factors are recruited to DNA damage sites via their association with chromatin, we examined the chromatin association of key DDR proteins following irradiation and the effect of nucleolin knockdown on the process. HR factors, Rad51, RPA34 and RPA70 in control cells accumulated into chromatin fraction at 4 hours after 5 Gy of irradiation, while they did not PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49245