Lack of NE translocation upon inhibition of actin cytoskeletal rearrangements. (A) Representative images of neutrophils pretreated with LatB, JSP, or the vehicle and stimulated with PMA for 60 minutes at 37°C. Neutrophils were stained for NE (magenta) and DNA (cyan; Hoechst). Images were acquired by using a Leica SP8 confocal microscope. (B) Quantification of NE translocation to the nucleus at t = 60 minutes (n = 3-4). (C) Schematic model of the phases of NETosis. Upon neutrophil activation, actin polymerization is induced. These dynamic actin rearrangements are necessary for NE to translocate to the nucleus where it can cleave nucleosomal histones.¹⁶ This promotes chromatin decondensation, whereupon the cytoplasmic milieu mixes with the nuclear material before the plasma membrane finally breaks down, resulting in NET release. Bars represent 25 μm (A).