S9.6 antibody but not GFP-dRNH1 binds to transfected dsRNA in human cells. (A) Representative confocal images showing HeLa cells transfected with ATTO-594 (red)-labeled ssDNA, ssRNA, dsRNA, or RNA–DNA hybrids and stained with S9.6 antibody (green). Inset images are magnified to show overlap of S9.6 signal and ATTO-594 foci. (B) Quantification of mean S9.6 intensities within individual oligonucleotide foci shown in A. (C) Same as in A, but with GFP-dRNH1 staining (green). (D) Quantification of mean GFP-dRNH1 intensities within individual oligonucleotide foci shown in C. (E) Representative confocal images of HeLa cells transfected with ATTO-594 (red) RNA–DNA hybrids and stained with GFP (green). 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 60 oligonucleotide foci scored per condition per experiment. ***, P ≤ 0.001 by Mann-Whitney U test; ns, P > 0.05. Scale bars are 10 microns; 5 microns for inset images. Inset images are magnified to show overlap of S9.6 or GFP-dRNH1 signal with ATTO-594 foci.