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. 2023 Sep 20;14:5832. doi: 10.1038/s41467-023-41391-7

Fig. 5. PAO1 induced NETosis is independent of ROS, GSDMD and GSDME.

Fig. 5

A–C Role of ROS in PMA stimulated, but not PAO1 infected peritoneal neutrophils. Representative time courses (A, B) and combined data from biological replicates (C). D-F IL-1β secretion (D) and NETosis (E) by peritoneal neutrophils incubated with reported GSDMD inhibitors necrosulfonamide (NSA), disulfiram, and LDC7559. NETosis was quantified by Sytox detection of extracellular DNA after 16 h (Area under the curve). Panel F is a representative time course. G–J Peritoneal neutrophils from C57BL/6 and Gsdmd-/- mice incubated 16 h with PMA, PAO1 or ∆pscD and extracellular DNA measured by Sytox. G representative time course, and H quantification of the area under the curve. I Representative images of citrullinated histone 3 (H3Cit) as an indicator of NETosis in PAO1 infected neutrophils (original and other images are in Fig. S4F). J Quantification of H3Cit+ neutrophils per high power field. K, L Representative time course and quantification of NETosis in C57BL/6 and Gsdme-/- mice. ** is <0.01, * is <0.05. Each data point represents one independent experiment (2-6 biological repeats). Error bars are mean +/- SEM. Statistical significance was assessed by 2-way ANOVA followed by Tukey’s post-test.