Figure 4.

The ankyrin repeats and the nuclease activity are essential for the function of ANKLE1. a) Schematic representation of a series of ANKLE1 truncations and the inactive mutant. b) U2OS cells expressing GFP‐tagged constructs were treated with or without 1 µg mL⁻¹ of doxycycline (Dox) that induces protein expression for 24 h. Cell extracts were analyzed by western blotting for the indicated proteins. c) Dox‐treated cells in b) were fixed for immunofluorescence. GFP (green), Aurora B (red) and DNA (blue) were visualized. d) Cells were treated with ICRF‐193 (100 × 10⁻⁹ m) for 16 h before fixed for immunofluorescence. 53BP1 (red), cyclin A (green), and DNA (blue) were visualized. e) Quantification of G1 cells (>1200 cells per condition) with more than four 53BP1 foci. Bars represent mean ± SD of n = 3 independent experiments. f) Clonogenic survival assay was carried out on cells treated with the indicated concentrations of cisplatin. Bars represent mean ± SD of n = 3 independent experiments. Scale bars, 10 µm. *p < 0.05; **p < 0.01; ns = not significant; statistical significance values were determined with unpaired two‐tailed t‐tests.