Fig. 1: Replacement of glucose with 2-DG inhibits the phosphorylation of GSK3 and increases GSK3 activity in a HK2-dependent manner.

a Rat1a cells or MEFs were incubated in glucose-free medium in the absence (−) or presence of 10 mM glucose (Glc), 2-DG or 5-TG. Immunoblots showing GSK3β phosphorylation, at the indicated time points after incubation. Bar graph shows densitometric quantification of pGSK3β/GSK3α/β ratio from three independent experiments in Rat1a cells (n = 3). Data are presented as the mean ± SEM. **p = 0.0063; n.s. p = 0.7392; one-way ANOVA was used to calculate significance. b Schematic depicting the structures of glucose (Glc), 2-DG, and 5-TG and their utilization by HK inside cells. Like Glc, 2-DG can be phosphorylated by HK2 but cannot be further metabolized except in the first step of the pentose phosphate pathway (PPP). 5-TG cannot be phosphorylated by HK2. c Rat1a cells were incubated in glucose-free medium in the presence of 10 mM 2-DG for the indicated durations. Cells were then harvested for immunoblotting to determine GSK3β phosphorylation and MCL1 levels. Bar graph shows quantification from two independent experiments. d MI5-4 CHO cells expressing either wild-type (WT) HK2, individual kinase-dead HK2 mutants (DA, SA) or empty vector (V) were incubated in glucose-free medium in the presence of 10 mM glucose (G) or 2-DG (D). After 2 h, cells were harvested and analyzed for immunoblotting. Bar graph shows relative densitometric quantification of pGSK3β/GSK3α/β ratio (WT, DA, SA). Results are the mean ± SEM of three independent experiments. *p = 0.0192; one-way ANOVA was used to calculate significance. e Representative immunoblot of three independent experiments showing the level of the MCL-1 protein after expression of WT or kinase-dead SA mutant HK2 in MI5-4 CHO cells. f Protein stability of MCL-1 in MI5-4 CHO cells and MI5-4 CHO cells expressing WT HK2 as measured after exposure to cycloheximide (CHX). Plot showing MCL1 protein half-life after quantification relative to β-actin. Data are presented as the mean ± SEM. *p < 0.05 (p = 0.0272 for 30 min and p = 0.0151 for 120 min), ****p < 0.0001 versus 0 h; two-way RM ANOVA test was performed to calculate the significance (n = 3 independent experiments). Uncropped blots are provided as a Supplementary Fig. 16. Source data are provided as a Source Data file.