DMSR depends on interphase centrosome maturation. (A) Quantitative RT-PCR and Western blot analysis show the siRNA efficiency of centrosome proteins NEDD1 and PCNT in U2OS cells. (B) Cell cycle stage was determined by flow cytometry in shcon or shPCNT U2OS cells (related to Fig. 5 B). Both shcon and shPCNT cells remained in G1 phase after IR treatment. (C) The efficiency of PLK1 inhibitor or p38 inhibitor was confirmed by pS216-CDC25C antibody or pThr180/Tyr182-p38 antibody. (D) Centrosome subdistal appendage protein Ninein and CEP170 obviously increased at the centrosomes after cells were exposed to IR in U2OS cells. Left: Immunofluorescence showed DSB-induced accumulation of Ninein or CEP170 in cells treated with 2 Gy IR. Right: Semiquantitative analysis of the relative signal intensity of subdistal appendage proteins at the centrosomes from control or IR-treated cells is shown. Scale bar, 20 µm. (E) Subdistal appendage protein recruitment at the centrosomes depends on DNA-PK kinase activity in U2OS cells. Right: DNA-PK inhibitor inhibited the IR-induced accumulation of Ninein and CEP170 at the centrosomes. Semiquantitative analysis of Ninein and CEP170 signal intensity at the centrosomes from control or DNA-PK inhibitor treated cells is shown. Left: IR-induced recruitment of Ninein and CEP170 at centrosomes requires 53BP1. Semiquantitative analysis of Ninein and CEP170 signal intensity at the centrosomes from control or 53BP1-depleted cells is shown in box plot (n > 50). ****, P < 0.0001; ***, P < 0.001; *, P < 0.05; ns, not significant. A.U., arbitrary units; UT, untreated; PI, propidium iodide.