CUX1 is necessary for DNA repair and focus formation. K562 cells were irradiated (6 Gy) and imaged 1 hour postirradiation. (A) Cells were imaged for antibodies targeting γH2AX and H3K27 methylation. H3K27me1/2/3 mean fluorescent intensity (MFI) was quantified within each γH2AX foci. (B) γH2AX foci size was quantified and broken into quintiles by size, with Q1 containing the smallest foci, and Q5 the largest. The size of individual γH2AX foci was determined using a custom ImageJ macro. EHMT2 intensity was quantified by MFI within each γH2AX foci quintile. (C) γH2AX foci size in gHPRT and gCUX1 cells (D) Mean number of γH2AX foci per cell following irradiation with and without UNC0642. (E) The intensity of total pATM (MFI) per nucleus was quantified after irradiation. (F) The intensity of pATM within γH2AX foci. (G) The intensity of 53BP1 within γH2AX foci. The median is shown, and a Mann-Whitney test was performed. (H-J) γH2AX was measured by flow cytometry in gHPRT and gCUX1 cells after 6 hours of mock treatment, etoposide (H), daunorubicin (I), or cisplatin (J). A Mann-Whitney test was performed. All plots are compiled from data derived from 3 independent biological replicates.