Immunofluorescence and microscopy
Mitotic abnormalities and G1-associated defects were analyzed as described previously.28, 29 In brief, for mitotic analysis, cells were seeded onto 22 × 22 mm glass coverslips in 6-well plates. After 18 hr, cells were treated with 3.5 μM RO3306 for 6 hr for the induction of a late G2 arrest and were subsequently released into fresh media. After 45 min, cells were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 in PBS for 20 min. For G1-associated defects, RO3306-treated cells were released into fresh media for 30 min. Prometaphase cells were shaken off and re-seeded onto glass slides coated with poly-l-lysine. After 4–6 hr, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 10 min. Fixed cells were incubated with antibodies specific to cyclin A (Santa Cruz Biotechnology, sc-596), 53BP1 (Santa Cruz Biotechnology, sc-515841), and PICH.30