Ab96883

Briefly, coverslips containing human primary pulmonary alveolar epithelial cells (HPAEpiCs) were washed twice with phosphate-buffered saline (PBS, pH 7.6) and fixed with fresh high-grade 4% paraformaldehyde (PFA) for 10 min. The PFA was aspirated and the coverslips were washed 4 times in PBS for 5 min each time. The coverslips were blocked with 1.5% Normal Goat Serum (NGS, Sigma Aldrich UK) for 2 h at room temperature. Primary antibody incubation was carried out for 2 h at room temperature or overnight at 4 °C with gentle agitation. HPAEpiCs were identified by using a Zonula Occludens primary antibody (Rb polyclonal anti ZO-1 – Abcam ab96587). ZO-1 antibody was used in a 1:100 dilution and the samples were washed in PBS for 5 × 5 min. The corresponding secondary antibody (goat anti-rabbit IgG Abcam ab96883) conjugated to Dylight^® (1:1000) was added to the cells for 2 h at room temperature with gentle agitation. Cells were washed in PBS for 5 × 5 min, and the coverslips were inverted onto a drop of mounting medium containing DAPI (Fluoroshield^TM Sigma Aldrich UK) on a microscope slide, and stored at 4 °C. The immunostained cells were viewed under fluorescence (Nikon Eclipse 80i fluorescent microscope) with the appropriate excitations for each fluorophore. All images were analysed using ImageJ-Win64.

We seeded 1 × 10⁴ cells on an 18-mm cover glass (GMA81-018, MATSUNAMI, Bellingham, WA, USA). After being treated with 2.5-μM Dac for 5 days, cells were fixed in 4% formaldehyde, washed and then permeabilized with 0.25% Triton X-100 in PBS for 10 minutes. Cells were subjected to staining with primary antibodies mouse anti-dsRNA mAb J2 (1:200, 10010200; SCICONS, Szirák, Hungary) for 2 h and rabbit anti-Tom20 pAb (1:100; sc11415, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h, secondary antibodies Alexa 488-conjugated donkey anti-mouse (1:500, ab96873, Abcam, Cambridge, UK) and Alexa 594-conjugated goat anti-rabbit (1:500, ab96883, Abcam, Cambridge, UK) for 1 h and DAPI (D1306, Thermo Fisher Scientific, Waltham, MA, USA). After washing twice with PBS, cover glass was mounted with Fluoromount-G^® (0100-01, SouthernBiotech, Birmingham, AL, USA). Super-resolution imaging was performed using a confocal microscope (LSM 980 with Airyscan 2, Zeiss, Oberkochen, Germany). The fluorescence signal intensity of dsRNA was captured under lower magnification (100×) of the Olympus FV10i confocal microscope. Quantification of the dsRNA signal was obtained in three fields containing at least 17 cells for each group from three independent experiments. The quantification of fluorescent signal representing dsRNA level was normalized to cell number.