Figure 1. Nuclear PKM2 is elevated in... | American Society of Hematology

Figure 1.

$Nuclear PKM2 is elevated in peripheral neutrophils after a stroke in humans and in WT mice and regulates neutrophil hyperactivation. (A) Top: schematic of experimental design. Bottom: western blot analysis of PKM2 in the cytosolic and nuclear fraction from the peripheral neutrophils isolated from the patients with acute ischemic stroke who underwent successful mechanical thrombectomy. The quantitative data for cytosolic and nuclear PKM2 intensity (normalized to the intensity of lamin-B1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown on the right. (B) Top: schematic of experimental design. Bottom: western blot analysis of PKM2 in the cytosolic and nuclear fraction from the peripheral neutrophils of male WT mice. The quantitative data for cytosolic and nuclear PKM2 intensity (normalized to the intensity of lamin-B1/GAPDH at each time point) are shown on the right. (C) Western blot analysis of PKM2 from neutrophils derived from the bone marrow of male mice. (D) Immunofluorescence analysis of NETs from peripheral neutrophils isolated 6 hours after reperfusion. Neutrophils were stimulated with a 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. (E) Inflammatory cytokines in peripheral neutrophils isolated 6 hours after reperfusion from each group as analyzed by enzyme-linked immunosorbent assay (ELISA). (F) Gene expression analysis for the neutrophils isolated 6 hours after reperfusion as analyzed by reverse transcriptase polymerase chain reaction. Data are from 2-way repeated measures analysis of variance (ANOVA) (Kruskal-Wallis test) followed by Fisher’s least significant difference (LSD) test; panels (A-B); or an unpaired Student t test (D-F). Data are mean ± standard error of the mean (SEM); n = 4-6 (A-B); n = 4-5 (D-E); n = 6 (F). AU, arbitrary units; HIF1-α, hypoxia-inducible factor 1-α; MCAO, middle cerebral artery occlusion; MMP9, matrix metallopeptidase 9; mRNA, messenger RNA; NS, not significant; TICI, thrombolysis in cerebral infarction.$

Nuclear PKM2 is elevated in peripheral neutrophils after a stroke in humans and in WT mice and regulates neutrophil hyperactivation. (A) Top: schematic of experimental design. Bottom: western blot analysis of PKM2 in the cytosolic and nuclear fraction from the peripheral neutrophils isolated from the patients with acute ischemic stroke who underwent successful mechanical thrombectomy. The quantitative data for cytosolic and nuclear PKM2 intensity (normalized to the intensity of lamin-B1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown on the right. (B) Top: schematic of experimental design. Bottom: western blot analysis of PKM2 in the cytosolic and nuclear fraction from the peripheral neutrophils of male WT mice. The quantitative data for cytosolic and nuclear PKM2 intensity (normalized to the intensity of lamin-B1/GAPDH at each time point) are shown on the right. (C) Western blot analysis of PKM2 from neutrophils derived from the bone marrow of male mice. (D) Immunofluorescence analysis of NETs from peripheral neutrophils isolated 6 hours after reperfusion. Neutrophils were stimulated with a 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. (E) Inflammatory cytokines in peripheral neutrophils isolated 6 hours after reperfusion from each group as analyzed by enzyme-linked immunosorbent assay (ELISA). (F) Gene expression analysis for the neutrophils isolated 6 hours after reperfusion as analyzed by reverse transcriptase polymerase chain reaction. Data are from 2-way repeated measures analysis of variance (ANOVA) (Kruskal-Wallis test) followed by Fisher’s least significant difference (LSD) test; panels (A-B); or an unpaired Student t test (D-F). Data are mean ± standard error of the mean (SEM); n = 4-6 (A-B); n = 4-5 (D-E); n = 6 (F). AU, arbitrary units; HIF1-α, hypoxia-inducible factor 1-α; MCAO, middle cerebral artery occlusion; MMP9, matrix metallopeptidase 9; mRNA, messenger RNA; NS, not significant; TICI, thrombolysis in cerebral infarction.

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