Fig. 4

EYA4 disturbs cell cycle progression and triggers cell cycle checkpoints. A Analysis of cell cycle distribution of asynchronized populations by flow cytometry. A representative cell cycle histogram is shown. The percentage of cells in G1, S or G2/M is plotted as mean ± SEM for three independent experiments, * P ≤ 0.05. B Percentage of HeLa FUCCI cells, transduced with EV control or EYA4 shRNAs, expressing each marker classified as in G1, G1-S transition or S-G2-M is plotted. C Representative images showing endomitosis (marked by white arrowhead) in HeLa FUCCI EYA4 depleted cells. D A schematic representation of double thymidine block for cell synchronization in early S-phase is shown. Cell synchronization was monitored by flow cytometry of PI-stained cells. The percentage of cells in G1/S or G2/M is plotted for three independent experiments. E Immunoblotting of cell cycle checkpoints proteins (cyclin E, p21, cyclin A, CDK2, and p27) in EYA4 depleted cells and controls, after double thymidine block (NS, non-synchronized population). β-Actin was used as the loading control. Western blots were repeated at least three times. Quantification was done on the represented immunoblot