Fig. 5

EYA4-depleted cells accumulate spontaneous replication stress. A Chk1 and Chk2 kinases in checkpoint control. A schematic representation of cell cycle checkpoints is shown (adapted from Collins and Garrett [44]). After double thymidine block, cells were released in early S-phase, activation of checkpoint kinase 1 and 2 (S345 and T68, respectively) and accumulation of spontaneous double-strand breaks in S-phase marked by γH2AX were followed by immunoblotting (NS, non-synchronized population). PCNA was used as the loading control. *Antibodies screened on the same membrane. Western blots were repeated at least three times. Quantification was done on the represented immunoblot. B EYA4-depleted cells show sensitivity to an ATR inhibitor, AZ20, using an MTT assay (mean ± SEM; n = 3). C Induction of CENP-F foci formation after exposure to γ-irradiation (4 Gy). Representative images (scale bar 10 μm) and quantification (n ≥ 100) in controls and cells depleted for EYA4 are shown