Figure 4

KAP1 phospho-Ser473 after DNA damage is Chk1- and Chk2-dependent. (a) Etoposide-induced KAP1 Ser473 phosphorylation is abolished by Chk1/Chk2 inhibition and reduced upon ATM inhibition. U2OS cells were untreated or treated with 5 μM etoposide (ETP) for 4 h in the presence or absence of KU55933 (ATMi), caffeine (Caff), or AZD7762 (AZD). (b) KAP1 phospho-Ser473 induction after 20 Gy of IR is abolished by AZD7762 (the drug was not removed during the recovery time). Chk2 phospho-Thr68 was used as readout of DNA damage and histone H3 phospho-Ser10 was used as readout for the G2/M checkpoint. (c) AZD7762 decreases KAP1 phospho-Ser473 on immunofluorescence; U2OS cells were treated as in (b). (d) KAP1 Ser473 is targeted by Chk1. U2OS cells were transfected with either siLuc, siChk1, siChk2, or both siChk1 and siChk2, then treated with 10 μM aphidicolin for 1 h. (e) KAP1 Ser473 is targeted by Chk2. U2OS cells were transfected as in (d) and treated as in (b). (f) KAP1 phospho-Ser473 is neither recruited nor excluded from laser-induced DNA-damage sites. Cells were fixed 5, 10 or 30 minutes after micro-irradiation.