PRC1 sequestration and loss of activity impact DDR factor recruitment to sites of damage. (A) Representative immunofluorescence images of 53BP1 signal (red) and H2AUb signal (green) in −dox, +dox, +eto, or +dox +eto iDUX4 cells (scale bar = 20 μm). White arrows indicate nuclei that have no H2AUb signal and no 53BP1 signal/recruitment to sites of damage. Asterisks indicate nuclei with H2AUb signal and 53BP1 foci. Images are representative from two independent experiments conducted on separate days. (B) Fraction nuclei with H2AUb signal type that contain 53BP1 foci. With H2AUb nuclear signal (positive) or lack H2AUb signal (negative) in +dox only or +dox +eto iDUX4 cells. Dots represent each independent experiment and N ≥ 100 nuclei per condition. (C) Combined RNA-FISH and immunofluorescence of HSATII RNA (green), γH2A.X (cyan), and 53BP1 (red) signal in +dox iDUX4 cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion (scale bar = 10 μm). Images are representative of two independent experiments conducted on separate days. (D) Fraction total nuclei with either γH2A.X signal only or both γH2A.X and 53BP1 signal in +dox cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion in nuclei that contained HSATII RNA foci (HSATII+). N ≥ 100 nuclei between all fields; dots indicate the average between fields. (B, D) Data represent means ± SD. Statistical differences between groups were assessed with one-way ANOVA Tukey’s multiple comparison test between each group and a control.