Figure 9.

Centrosome integrity is important to DSB mobility and NHEJ repair. (A) Examples of traces of GFP–53BP1TD foci in shcon or shPCNT G1 HeLa cells. Images were collected in cells expressing GFP–53BP1TD (residues 1220–1711) 1 h after IR (see also Videos 10 and 11). Scale bars, 10 µm for labeled panel, 2 µm for magnified images. (B) Nocodazole (Noco) treatment or depletion of PCNT or NEDD1 decreased DSB mobility. eMSD of the GFP–53BP1TD foci is shown in indicated conditions and for every time point. Values are shown as mean ± SEM. The number of traces were pooled from three independent experiments. (C) Depletion of PCNT delayed NHEJ as determined by γH2AX foci in different time points after IR. Left: Immunofluorescence showed γH2AX foci in scramble shRNA- or shPCNT-treated G1 cells. Right: Quantitation of cells with γH2AX foci in control or PCNT-depleted G1 cells at indicated time points (n > 50). Scale bar, 20 µm. (D) The kinetics of DNA damage repair in IR-treated G1 HeLa cells were determined by 53BP1 foci in shcon, shPNCT, or shNEDD1 cells (n > 100). (E) RPA2 foci induced by IR increased in PCNT-depleted G1 HeLa cells. Left: RPA2 foci in control cells or PCNT-depleted cells. Right: Quantitation of cells with RPA2 foci in control cells or PCNT-depleted cells (n > 50). Scale bar, 20 µm. (F) Upper: Representative images showed chromosome breaks in shcon or shPCNT HeLa cells after IR. Arrow indicates chromosome breaks. Lower: Quantitation of chromosome breaks per cell in shcon or shPCNT cells with or without IR treatment (n > 30). **, P < 0.01; *, P < 0.05. UT, untreated.