Fig. 6

The impact of EYA4 on replication fork progression. A EYA4-depleted cells show sensitivity to hydroxyurea using an MTT assay (mean ± SEM; n = 3). B DNA synthesis was assessed by EdU incorporation. Representative images (scale bar 10 μm) and percentage of EdU incorporation in the presence of 4 mM hydroxyurea are shown for three independent experiments. C-D Accumulation of hydroxyurea-induced γH2AX foci formation. Representative images (scale bar 10 μm), quantification (mean ± SEM; n ≥ 350), and more than 10 foci per nucleus are shown for 4 mM HU treatment for 2 h followed by 2 h release (C) or 16 h HU treatment followed by 18 h release (D). E A representative DNA fiber image is shown for each genetic condition. Replication fork speed (kb/min) is shown for empty vector control, EYA4-depleted cells and EYA4 phosphatase mutant cells (mean ± SEM; a minimum of 150 forks was scored in two independent experiments yielding similar results. Statistical analysis: unpaired t-test). For all panels * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. F Model: Over-expression of EYA4 leads to an aggressive and invasive breast cancer phenotype. EYA4 has a protective role in cells against replication stress, triggering the activation of the ATR pathway and cell cycle arrest