Quantifying DNA Damage by Comet Assay

U2OS cells were treated with G9a inhibitor (UNC0638, 1 µM) or ATM inhibitor (KU55933 [16] (link), 10 µM) for 24 h, while HCT116 p53 WT/KO cells were treated for 4 days. For siRNA-transfected cells, assays were performed 72 h after transfection. Neutral comet assay were performed with the damaging agent phleomycin, as previously reported [17] (link). Briefly, an appropriate number of cells were mixed with low-melting Agarose (Trevigen) and bound on GelBond film (Lonza). Samples were lysed and electrophoresed at 35 V for 7 min. The samples were fixed, dried and stained with SYBR Green I (Invitrogen). Images were taken with an IX71 fluorescent microscope (Olympus) using Cell^F software (Olympus). Tail moments were quantified using CometScore software (TriTek). Means of tail moments of at least 50 cells were measured per condition.