Extended Data Fig. 4: Identification and functional analysis of Hexokinase 1 T259 O-GlcNAcylation Site.

a, Tandem mass spectra showing O-GlcNAc on peptides derived from human Hexokinase 1. Data were acquired using HCD fragmentation and prominent y and b-type ions are labeled. Blue arrow indicates the O-GlcNAc modified threonine (T). Bottom figure demonstrating the survey scan and prominent y/b-type ions. b–e, Quantitative analysis of HK1 co-localization to measure the percentage of mitochondrial HK1 intensity in HEK293T cells, cultured in 5 mM glucose containing media. (b and d) Representative images of HEK293T cells expressing WT-HK1-GFP or T259A-HK1-GFP (green) and Mito-DsRed (magenta) with or without OGT overexpression and Thiamet-G treatments. Scale bars represent 10 μm (c and e) WT and T259A HK1 intensity on mitochondria, percentage of total WT and T259A HK1 on mitochondria and the Pearson’s correlation coefficient (R value) for each condition. Data are presented as violin plots with individual data points and associated p-values. n = 9 cells, three biological replicas (two-tailed Mann-Whitney U test). f, Representative images of hippocampal neurons expressing HK1-shRNA, WT-HK1-GFP, and the O-GlcNAc mutant T259A HK1-GFP (T259-HK1-GFP) are shown in green, along with Mito-DsRed in magenta. These images were stained with an HK1 antibody (in cyan) to visualize the total HK1 expression. Scale bar represents 5 µm. g, Quantification of HK1 expression levels was performed retrospectively for all experiments by analyzing the anti-HK1 staining to ensure consistent endogenous HK1 levels throughout all experiments. All values are shown as mean ± SEM (one-way ANOVA with post hoc Tukey’s multiple comparison test). h, Quantification of the size of the mitochondria along the axons as depicted in Fig. 4g. n = 81–86 mitochondria from 10–13 axons from three biological replica (one-way ANOVA with post hoc Kruskal-Wallis multiple comparison test).

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