Cells were grown on chamber slides (Nunc LabTek Chamber Slide, ThermoFisher Scientific), treated, and fixed in cold acetone (for HMGB1 staining) or 4% paraformaldehyde (for NF-κB p65 staining). Cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich Corp.) in PBS (HMGB1) or cold methanol (NF-κB p65), followed by blocking with 15% goat or donkey serum (Abcam, Cambridge, MA, USA). Slides were incubated overnight with primary antibodies: rabbit anti-human/mouse HMGB1 (Abcam) or rabbit anti-human NF-κB p65 (Abcam) at 4°C, washed with PBS, and incubated for 1 hour with secondary antibodies (Alexa Fluor 488 goat or donkey anti-rabbit IgG; Invitrogen, Life Technologies, Carlsbad, CA, USA) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Biotium, Hayward, CA, USA). Cover slips were then mounted with Airvol mounting media (courtesy of Alan Burns, PhD, University of Houston, College of Optometry, Houston, TX, USA) and slides imaged with the DeltaVision Imaging System (GE Healthcare, Issaquah, WA, USA).