BRCA1 and SETX depletion leads to increases in RNA–DNA hybrids, detectable by GFP-dRNH1. (A) Representative confocal images of HeLa cells transfected with control or BRCA1-targeting siRNAs. After fixation, coverslips were treated with the following enzymes: none (Mock); RNase H (H); RNase T1 and RNase III combined (T1 + III); RNase H, RNase T1, and RNase III combined (T1 + III + H). GFP-dRNH1 signal is shown in green, DAPI in blue. (B) Quantification of mean nuclear GFP-dRNH1 intensities for the conditions shown in A. (C) Same as in A but with transfection of control or SETX-targeting siRNAs. (D) Quantification of mean nuclear GFP-dRNH1 intensities for the conditions shown in C. Box plots show median (box central line), 25% and 75% percentiles (box edges), and minimum and maximum values (whiskers). Data are combined from three biological replicates (n = 3), with at least 80 nuclei scored per condition per experiment. ***, P ≤ 0.001 (Mann-Whitney U test). Scale bars are 10 microns.