13 mm coverslips

Acinar cells were isolated and seeded onto 13 mm coverslips (Thermo Fisher Scientific, Waltham, MA, USA). The cells were treated with 250 µM hydrogen peroxide (7.5 min) with or without 10–30 µM TCT pretreatment (1 h). Coverslips were incubated in methanol for 20 min at −20 °C, washed with phosphate-buffered saline (PBS), and lysed with 10% Triton-X in PBS. After blocking in 1% bovine serum albumin (BSA) in PBS at room temperature, cells were incubated with a mouse monoclonal poly(ADP-ribose) (PAR) specific antibody (purified in-house from the supernatant of the 10H hybridoma; isotype IgG3) overnight at 4 °C. On the following day, cells were incubated with a goat anti-mouse Alexa633-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) (1:1000) and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) (1:2000) at room temperature. The coverslips were viewed with an SP8 Leica confocal microscope. The intensity of the PAR signal was analyzed with ImageJ software.

For IF staining cells were seeded onto 13 mm coverslips (Thermo Fischer Scientific) in 24-well plates. The reptarenavirus NP-transfected cells were PBS washed twice (at three dpt) and fixed with 4% PFA (in PBS) for 10–30 min at RT. After a PBS wash, cells were permeabilized and blocked (0.25% TX-100 and 0.5% (w/v) BSA in PBS) for 5–10 min at RT, and washed twice with PBS. The cells were incubated with the primary antibodies (overnight at 4 °C or 1 h at RT), washed 3–5 times with PBS, incubated 1 h at RT with the secondary antibodies, washed 3–5 times with PBS, incubated 15 min at RT with DAPI (Novus Biologicals, 1 µg/µl, diluted 1:10,000 in PBS), and washed twice with milli-Q water prior to mounting with FluoreGuard mounting medium (Scytek Laboratories). All primary and secondary antibodies were diluted in Dako REAL antibody diluent (Agilent technologies). Images were captured and analyzed using a Nikon Eclipse TI microscope with NIS-Elements Microscope Imaging Software (Nikon).