ML265 treatment significantly reduces PKM2 nuclear translocation and neutrophil hyperactivation after acute ischemic stroke. (A) Schematic of experimental design. (B) Western blot analysis of PKM2 in the cytosolic and nuclear fraction from the peripheral neutrophils isolated 6 hours after reperfusion in mice treated with ML265 at the indicated doses. The quantitative data of cytosolic and nuclear PKM2 intensity (normalized to the intensity of lamin-B1 or GAPDH at each time point) are shown on the right. (C) TNF-α, IL-1β, and IL-6 levels in neutrophils isolated 6 hours after reperfusion from each group as analyzed by ELISA. (D) Immunofluorescence analysis of NETs from the neutrophils isolated 6 hours after reperfusion. Neutrophils were stimulated with the suboptimal concentration of PMA (10 ng/mL), and NETs were visualized by using SYTOX Green stain. Scale bars, 100 µm. Quantification is shown on the right. Data are from male WT mice and are mean ± SEM; n = 3 (B); n = 4 (C); n = 5 (D). Data are from 2-way repeated measures ANOVA (Kruskal-Wallis test) followed by Fisher’s LSD test (B), or an unpaired Student t test (C,D).