Figure 3.

RIF1 Deficiency Suppresses Proliferation and Genome Instability Defects in BRCA1-Depleted Cells

(A) HeLa cells were treated with the indicated siRNA for 48 hr. Cells were then pulse-labeled with EdU (5 min, 40 μM) immediately before irradiation (5 Gy). One hour following IR, cells were fixed and permeabilized before fluorescent labeling of EdU and counterstaining with indicated antisera. Representative projection images of whole-nuclei 0.5 μm confocal z series are presented.

(B) Enlarged S phase images of RIF1 IRIF from (A) and automated quantification of the intensity of RIF1 IRIF in HeLa cells subjected to control or BRCA1-targeting siRNA. n > 140 cells per condition; ^∗∗∗p < 0.0001, ns¹ p = 0.514, ns² p = 0.1172, ns³ p = 0.1390, Mann-Whitney test.

(C) Western blot shows comparable BRCA1-depletion efficiency between MEF lines selected for expression of indicated shRNA constructs. C, control shRNA vector; 1, Brca1 shRNA vector 1; 2, Brca1 shRNA vector 2.

(D) Radial chromosomes were scored in metaphases prepared from WT, Rif1^−/−, and 53Bp1^−/− cell lines expressing the indicated shRNA following 16 hr control or olaparib (1 μM) treatments. n = 50 metaphase per condition. Error bars are ± SEM. See also Figure S4.