Confocal microscopy and image analysis.

A549 cells were seeded in 24-well plates on poly-l-lysine-coated coverslips (Neuvitro) and were either transfected with IAV HA-encoding plasmid or infected with influenza A/Puerto Rico/8/34 (H1N1) virus. Cells were fixed with 4% paraformaldehyde (Alfa Aesar) and then permeabilized, blocked with 10% FBS and 1% BSA for 1 h, and incubated with primary antibodies, i.e., anti-IAV HA (Santa Cruz) or anti-Myc tag (Cell Signaling Technology) antibody and anti-PARP1 antibody (Cell Signaling Technology) overnight at appropriate concentrations in 1% BSA. After washing, samples were incubated with the appropriate secondary antibodies, such as Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) or Alexa Fluor 546 goat anti-rabbit antibody (Invitrogen), for 1 h and then washed. Cell nuclei were stained using Draq5 according to the manufacturer’s instructions (Thermo Fisher) and then mounted on glass slides with Prolong gold antifade mountant (Thermo Fisher). Confocal images of thousands of cells were acquired at the University of Missouri Molecular Cytology Core facility using a Leica SP8 TCP confocal microscope. For quantification purposes, imaging was performed using a 40× objective, capturing 8 to 10 random images per condition per experiment. Quantification was performed using Fiji (48) of ImageJ (49). Levels of cytoplasmic PARP1 were determined by using the ImageJ Intensity Ratio Nuclei Cytoplasm Tool (50, 51), where Draq5 was used as the nuclear stain. The threshold, select area, and ROI (region of interest) manager functions of ImageJ were used to restrict measurements of the Intensity Ratio Nuclei Cytoplasm Tool to cells that only expressed HA. The Manders colocalization coefficient (MC) between HA and PARP1 was determined using the ImageJ plugin JACoP (52).