Fig. 5: PRL2 regulates NET formation through the Rac-ROS pathway.

a Wildtype (WT) and PRL2 knockout (KO) bone marrow derived neutrophils (BMNs) were stimulated with phorbol myristate acetate (PMA). Reactive oxygen species (ROS) production was measured by chemiluminescence assay. ROS kinetic plots and averaged area under the curve (AUC) are shown. b WT and PRL2 KO neutrophils were treated with PMA, iRBCs and hemin, ROS production was measured by fluorescent staining. a, b Data were pooled from four independent experiments. BMNs were isolated from different mice (n = 4 per group). c Quantification of neutrophil extracellular traps (NETs) released by WT and PRL2 KO neutrophils that stimulated with PMA, hemin and iRBCs. Cells were pretreated with NAC or not. Data were pooled from three independent experiments. BMNs were isolated from different mice (n = 3 per group). d WT and PRL2 KO neutrophils were treated with PMA for the indicated times. Cell lysates were subjected to pull-down using PAK GST beads. Lysates from pull-down and total lysates were subjected to SDS-PAGE followed by immunoblot assay with anti-Rac, anti-PRL2 and anti-GAPDH antibodies. The experiments were repeated two times with similar results, and the representative immunoblot images were shown. e Representative immunofluorescence images of WT and PRL2 KO neutrophils after PMA stimulation. The experiments were repeated three times with similar results. DNA is stained in blue (DAPI), and GTP-Rac is stained in green. Scale bar, 5 μm. f Relative fluorescence intensity (RFI) of GTP-Rac from WT or KO cells in (e) was determined (n = 26 cells per group). g Quantification of NETs released by WT and PRL2 KO neutrophils that stimulated with PMA, hemin and iRBCs. Cells were pretreated with NSC23766 or not. Data were pooled from three independent experiments. BMNs were from different mice (n = 3 per group). Data are presented as the mean ± SEM or in violin plots showing the median and interquartile range. p values were calculated by two-way ANOVA with Tukey’s multiple testing (a right, b, c, f, g) and shown in the figures. Source data are provided as a Source Data file.