WT-MEF were plated 24 hours before transfection with Cyanin3-labeled RNA:DNA hybrids (2 μg/ml) in Opti-MEM using the JetPrime reagent, according to the manufacturer’s instructions. Two hours after transfection, cells were detached with 0.5 mM EDTA in PBS and plated on fibronectin-treated coverslips. Four hours later, cells were fixed with 4% PFA in PBS, and permeabilization was performed with 0.1% Triton X-100 in PBS for 5 min at room temperature (RT). After blocking in PBS containing 0.1% Tween (PBS-T) and 5% BSA for 30 min at RT, cells were incubated overnight with the S9.6 antibody at 1/200 dilution in PBS-T, 5% BSA.RNaseH treatment before primary antibody incubation was performed, when indicated, with 60 U of RNaseH (Ambion) in RNaseH buffer in a humid chamber overnight at 30°C. Secondary antibody incubation was performed for 1 hour at RT. Cytoskeleton was stained with Actin Green 488 ReadyProbes reagent (Molecular Probes by Life Technologies), and the nucleus with 4′,6-diamidino-2-phenylindole. Vectashield reagent was used to mount the coverslips. Z-stacks were acquired by a Apotome Z2 microscope by Zeiss with ZEN (blue edition) software, and images were processed with Fiji.