Figure 7.
ATAD5 depletion reduces the association of RNA helicases with normal and stalled replication forks. (A) HEK293T cells were labeled with EdU for 20 min and then treated with 10 μM thymidine for indicated times. Whole cell extracts were prepared for an iPOND EdU-thymidine chase assay. (B) HEK293T cells were transfected with ATAD5 siRNA for 48 h then whole cell extracts were prepared for an iPOND assay. (C–F) U2OS-TetOn-ATAD5 cell lines were transfected with ATAD5 siRNAs and treated with doxycycline to express wild type ATAD5 (ATAD5WT) or UAF1 interaction defective mutant ATAD5 (ATAD5ΔUAF1). After 48 h, cells were fixed for a SIRF assay on DDX21 (C, D) or DDX5 (E, F). Hydroxyurea (HU) was added at a final concentration of 2 mM for 3 h before fixation. (C, E) Representative SIRF images. (D, F) Number of nuclear SIRF signal foci were counted and plotted. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; **** P < 0.001, *** P < 0.005, **, P < 0.01, * P < 0.05 and ns, not significant.