Increased S9.6 immunofluorescent signal upon BRCA1 and SETX depletion is due to binding to dsRNA. (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). S9.6 signal is shown in green, DAPI in blue. (B) Quantification of mean nuclear S9.6 intensities for the conditions shown in B. Box plots show median (box central line), 25% and 75% percentiles (box edges), and minimum and maximum values (whiskers). (C) Levels of BRCA1 analyzed by Western blotting. Tubulin serves as a loading control. (D) Same as in A, but with transfection of control or SETX-targeting siRNAs. (E) Quantification of mean nuclear S9.6 intensities for the conditions shown in D. Box plots show median (box central line), 25% and 75% percentiles (box edges), and 10% and 90% percentiles (whiskers). (F) Protein levels of SETX as analyzed by Western blotting. Tubulin serves as a loading control. Data are combined from three biological replicates (n = 3), with at least 120 nuclei scored per condition per experiment. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (by Mann-Whitney U test); ns, P > 0.05. Scale bars are 10 microns. Mw, molecular weight in kDa.