Fig. 5. G6P accumulation inhibits CK2-mediated MLX phosphorylation and the binding of ChREBP-MLX tetramer on the ChoRE.

a. MLX phosphorylation in 293T cells starved for glucose and serum. CALX serves as a loading control. t-test, ****p<0.0001. N=6.

b-c. MLX phosphorylation in 293T cells starved for glucose and serum overnight and re-fed with 25 mM glucose. One-way ANOVA, **p<0.01, ****p<0.0001. N=4.

d-e. MLX phosphorylation in WAT from overnight fasted or overnight fasted and 3-hour re-fed mice. One-way ANOVA, ****p<0.0001. n=6.

f-g. MLX phosphorylation in 293T cells starved for glucose and re-fed with 25 mM 2DG. One-way ANOVA, ****p<0.0001. N=4.

h. ChREBP-MLX luciferase reporter activity in 293T cells expressing ChREBP, HK2 and MLX-WT. Cells starved for glucose and re-fed with 25 mM glucose or 25 mM 2DG for 3 hours. One-way ANOVA ****p<0.0001, ns=not significant. N=3.

i. In vitro CK2-mediated MLX phosphorylation (thiophosphate) in the presence of different concentrations of glucose-6-phosphate (G6P) or glucose. One-way ANOVA, **p<0.01,***p<0.001. N=4.

j. Binding of ChREBP and/or MLX to single E-box, perfect-ChoRE or PK-ChoRE probes. ChREBP and MLX were purified from 293T cells treated with or without 25 mM 2DG for 1 hour.

k-l. Quantification in j. t-test, *p<0.05, ****p<0.0001. N=3–4.