Figure 4.
Limiting PKM2 dimerization inhibits NETosis in vitro and reduces SNAP23 phosphorylation and α-granule exocytosis in platelets. (A) NETosis assay was performed by stimulating bone marrow–derived WT neutrophils with the releasates collected from ML265 (100 μM)- or vehicle-pretreated, thrombin-stimulated (0.1 U/mL) platelets. Representative microphotographs of NET in lower magnification (20×) and higher magnification (100×) stained with Sytox green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue) are shown on the left; higher magnification images showing NET from the boxed regions (scale bar for 20× image, 50 μm; scale bar for 100× image, 10 μm). Quantification of the percentage of cells releasing NET in lower magnification is shown on the right (n =5 per group). Values are mean ± SEM; unpaired Student t test. (B) WT platelets pretreated with vehicle or ML265 (50 and 100 μM; 10 minutes) were stimulated with thrombin (0.1 U/mL; 2 minutes), and the level of SNAP-23 phosphorylation was measured by western blot. The upper panel shows a representative western blot, and the lower panel shows a densitometry analysis of western blots (n = 4 per group). Values are mean ± SEM; 1-way ANOVA with Tukey multiple comparisons test. (C) Platelets pretreated with vehicle or ML265 (50 and 100 μM; 10 minutes) were stimulated with thrombin (0.1 U/mL; 2 minutes) and centrifuged for 3 minutes; the level of PF4 was measured in the supernatant by western blot. The upper panel shows a representative western blot, and the lower panel shows Ponceau S staining (n = 4 per group). Values are mean ± SEM; 1-way ANOVA with Tukey multiple comparisons test. Rest, resting platelets; Veh, vehicle.

Limiting PKM2 dimerization inhibits NETosis in vitro and reduces SNAP23 phosphorylation and α-granule exocytosis in platelets. (A) NETosis assay was performed by stimulating bone marrow–derived WT neutrophils with the releasates collected from ML265 (100 μM)- or vehicle-pretreated, thrombin-stimulated (0.1 U/mL) platelets. Representative microphotographs of NET in lower magnification (20×) and higher magnification (100×) stained with Sytox green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue) are shown on the left; higher magnification images showing NET from the boxed regions (scale bar for 20× image, 50 μm; scale bar for 100× image, 10 μm). Quantification of the percentage of cells releasing NET in lower magnification is shown on the right (n =5 per group). Values are mean ± SEM; unpaired Student t test. (B) WT platelets pretreated with vehicle or ML265 (50 and 100 μM; 10 minutes) were stimulated with thrombin (0.1 U/mL; 2 minutes), and the level of SNAP-23 phosphorylation was measured by western blot. The upper panel shows a representative western blot, and the lower panel shows a densitometry analysis of western blots (n = 4 per group). Values are mean ± SEM; 1-way ANOVA with Tukey multiple comparisons test. (C) Platelets pretreated with vehicle or ML265 (50 and 100 μM; 10 minutes) were stimulated with thrombin (0.1 U/mL; 2 minutes) and centrifuged for 3 minutes; the level of PF4 was measured in the supernatant by western blot. The upper panel shows a representative western blot, and the lower panel shows Ponceau S staining (n = 4 per group). Values are mean ± SEM; 1-way ANOVA with Tukey multiple comparisons test. Rest, resting platelets; Veh, vehicle.

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