Figure 3.

E2-induced DSB repair. (A) MELN cells were grown in phenol red-free medium supplemented with 10% charcoal–dextran-stripped FBS for 3 days. After pretreatment with 100 nM of E2 for 20 h, cells were left untreated or treated with IR (10 Gy). Cells were fixed for immunofluorescent staining with the γ-H2AX antibody (Ser 139) as a marker for DSBs. Foci were counted in at least 50 cells per condition. Error bars represent the s.d. values from at least three independent experiments. Differences were statistically significant at the P<0.05 level. (B) MELN cells were grown as above. After ±pretreatment with 100 nM of E2 for 20 h, cells were exposed to IR (20 Gy) and incubated for different times. The distribution of tail moments among a population of 100 cells for each time point was plotted. Error bars represent the s.d. values from at least three independent experiments. Representative images are shown additionally. Scale bars, 100 μm. (C) MELN cells were treated as in (A) followed by immunoblotting with specific antibodies as indicated. The asterisk represents an unspecific band recognized by the Chk2 antibody. (D) MELN cells were grown in phenol red-free medium supplemented with 10% charcoal–dextran-stripped FBS for 3 days. After pretreatment with 5 μM of NU7026 and 100 nM of E2 for 20 h, cells were treated with IR (10 Gy) and incubated for 2 h. Cell lysates were assayed for expression of proteins with the indicated antibodies. β-Actin was used as a loading control. ATM, ataxia teleangiectasia mutated; Chk2, checkpoint kinase 2; DSB, double-strand break; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; E2, oestrogen; ERα, oestrogen receptor-α; FBS, fetal bovine serum; γ-H2AX, phosphorylated histone H2AX; IB, immunoblotting; IR, ionizing radiation; U, untreated.