Fig. 1: ATAD5 promotes replication fork restart at stalled replication forks.

a The scheme for cell cycle arrest (Noco-APH condition). U2OS cells were arrested at the G1/S boundary and then released from arrest in normal media for 4 h. Human ATAD5 small interfering (si) RNA was transfected when cells were re-seeded after shaking-off. b U2OS cells released from arrest for 4 h were collected for cell cycle analysis. Asynchronous cells were transfected with ATAD5 siRNA for 48 h before cell collection. Under both conditions, cells were pulse-labeled with 5-ethynyl-2ʹ-deoxyuridine (EdU) for 30 min before cell collection. c, d U2OS or HeLa cells were transfected with ATAD5 siRNA under the Noco-APH condition. e U2OS cells expressing ATAD5^AID were pre-treated with auxin. c–e After depletion of ATAD5, cells were analyzed using a DNA combing assay. c Representative images of replication tract. Each asterisk (*) indicates a stalled replication fork. d, e The percentages of stalled forks are displayed. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. f U2OS cells were transfected with ATAD5 siRNA under the Noco-APH condition. Then, cells were subjected for a DNA combing assay. The length of l-dU-labeled DNA track was measured. g–j U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were labeled with 5-bromo-2ʹ-deoxyuridine (BrdU) for 10 min and incubated with 2 mM HU for 4 h before fixation. g, h, j Single-stranded DNA with BrdU exposed was visualized by staining with an anti-BrdU antibody under native non-denaturing conditions. i Total BrdU in DNA was detected under denaturing conditions as a control. g Representative images of anti-BrdU antibody staining. Scale bar: 20 μm. h, i The intensity of BrdU staining was quantified from ~200 cells and plotted. Three independent experiments were performed and one representative result was displayed. j 50 μM mirin was simultaneously administered with 2 mM HU for 4 h before fixation. f, h, i, j Boxes indicate median and interquartile ranges and whiskers indicate the 5th–95th percentile. Statistical analysis: Mann–Whitney U test; ****p < 0.0001, ***p < 0.001, **p < 0.01, n.s., not significant.