Extended Data Fig. 10: Determination of the HK2 relevance for microglia phagocytosis.
From: Early life high fructose impairs microglial phagocytosis and neurodevelopment
(a) Inhibition of HK2 activity rescues high fructose-induced suppression of microglia phagocytic activity. Microglia were plated and conditioned as in Fig. 2c, but with the inclusion of the HK2-specific inhibitor 3-BP. Conditioned microglia were cultured with CypHer5E-labeled apoptotic neurons for 1 h at a 0.5:1 target-to-phagocyte ratio, then isolated and analyzed for phagocytosis via flow cytometry. Shown is the phagocytic index (percent phagocytosis in experimental microglia divided by percent phagocytosis in control microglia) calculated from three independent experiments. Data are shown as mean ± SEM. Significant was calculated by two-way ANOVA with Tukey’s multiple comparisons test. **p < .01. (b) High fructose enhances HK2 expression. Microglia were treated identically to cells in (a), but pooled from multiple independent wells for western blot analysis. Data is representative of two independent experiments. For gel source data, see Supplementary Fig. 1. (c, d) High fructose increases mitochondrial HK2 colocalization in a GLUT5-dependent manner. Primary microglia from WT and KO mice were cultured with 5 mM glucose or 5 mM glucose and 5 mM fructose with mannitol as a control for osmolarity. Mitotracker Red CMXRos-labeled microglia were cultured with CypHer5E-labeled apoptotic neurons at a 1:1 ratio for 30 min, then fixed and analyzed via confocal microscopy. (c) Representative images were taken using confocal microscopy (63x oil immersion objective, 7 μm thick image stacks with 0.5 μm step-size), and max-intensity z-projections were used to analyze colocalization between HK2 and Mitotracker. (d) Pearson’s coefficients were calculated in Fiji, with 1 denoting perfect colocalization and 0 denoting no colocalization. Data shown is representative of two independent experiments, with n = 37 to 40 cells per condition. Scale bar, 10μm.
