Fig. 4: ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress.

a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5^AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.