Pharmacological inhibition of actin glutathionylation prevents DNA release. (A) Immunoblotting. Actin glutathionylation in activated mouse and human neutrophils is dependent on NADPH oxidase activation. Human and mouse neutrophils were analyzed after short-term stimulation (total 35 min) with the indicated triggers. C5a activation was performed in a time-dependent manner. No glutathionylated actin at the expected size (42 kD) was detected in Nox2^−/− mouse neutrophils or in 50-µM-DPI–pretreated human neutrophils. The ratio of glutathionylated actin to total actin is shown as actin glutathionylation in the bar graph (right). Data are representative of three independent experiments. (B) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of control human neutrophils with the indicated triggers in the presence and absence of the inhibitors as designated: 50 µM Na-arsenite and 2 µM CdCl₂. Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. *, P < 0.0242; **, P < 0.0074; ***, P < 0.001. n = 3. Bars, 10 µm.