Fig. 3. Fibrillarin (FBL) promotes the recruitment of DNA repair protein RAD51 to DNA damage sites. (a) FBL deficiency has no effect on cell cycle distribution. BrdU incorporation assays were performed to assess cell cycle distribution as described in the Materials and Methods section. (b) FBL silencing in U2OS cells impairs RAD51 foci formation at DNA damage sites, while replication protein A2 (RPA2) recruitment remains unaffected. Cells were treated with 1 μmol/L camptothecin (CPT) for 3 h and subjected to RAD51 and RPA2 immunofluorescence. (c) Quantification of RAD51 and RPA2 foci formation. (d) Rescue of RAD51 recruitment at DNA damage sites by re-expressing wild-type FBL in FBL-depleted U2OS cells. Cells stably expressing an empty vector or hemagglutinin (HA) and flag (HF)‍-tagged wild-type FBL (HF-FBL) were transfected with the indicated siRNAs, then treated with 1 μmol/L CPT for 3 h and subjected to RAD51 and RPA2 immunofluorescence. (e) Quantification of RAD51 and RPA2 foci formation. Data are presented as mean±standard deviation (SD), n=3. ^*** P<0.001; ns, not significant (Student's two-tailed t-test). BrdU: bromodeoxyuridine; DAPI: 4',6-diamidino-2-phenylindole; PI: propidium iodide; PE: P-phycoerythrin; siCon: control small interfering RNA; siFBL: FBL small interfering RNA; siR: small interfering RNA-resistant.