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. 2024 Feb 29;15:1879. doi: 10.1038/s41467-024-45548-w

Fig. 5. Hexokinase 2 is a part of an amplification loop linking glucose mediated ChREBP activation with enhanced PI3K/AKT signaling.

Fig. 5

a–d C57BL6/J male mice were injected with either GFP or ChREBP overexpressing adenovirus. Simultaneously, P85α expression was also inhibited through adenoviral-mediated shRNA delivery. Mice were study 3 weeks later. a Representative in vivo bioluminescence imaging depicting ChREBP activity on ChREBP-regulated reporter construct (ChoRE-luc) (n = 6 biologically independent mice per group). b Representative Western blot analysis of ChREBP sub-cellular localization in response to P85α silencing (n = 6 biologically independent mice per group). c Measurement of HK2 activity (n = 6 biologically independent mice per group). d Measurement of G6P concentration (n = 6 biologically independent mice per group). e–h FLAG tagged WT or HA tagged Δ196 isoforms of ChREBP were overexpressed in Huh7 cells. Cells were then treated with 100 nM of MK-2206 for 24h. e Representative Western blot depicting ChREBP cellular localization and PI3K/AKT signaling in response to MK-2206 treatment (n = 3 independent experiments). f Measurement of G6P concentration (n = 3 independent experiments). g LPK expression relative to the TBP gene expression (n = 3 independent experiments). h Model of the amplification loop linking glucose mediated ChREBP activation with enhanced PI3K/AKT signaling. i–l HK2 was overexpressed in Huh7 cells using HK2 expressing adenovirus. After HK2 overexpression (24 h), cells were then treated with 100 nM of MK-2206 for 24 h. i Representative Western blot showing the effect of HK2 expression on PI3K/AKT signaling (n = 6 independent experiments). j Measurement of G6P concentration in Huh7 cells (n = 6 independent experiments). k Measurement of ChREBP transcriptional activity on ChoRE-luc reporter construct (n = 6 independent experiments). l Representative clonogenic assays shown (n = 6 independent experiments). m–p ChREBP expression was inhibited in HK2 overexpressing Huh7 cells. m Representative Western blot depicting the activity of the PI3K/AKT signaling pathway in response to ChREBP silencing (n = 6 independent experiments). n Measurement of G6P concentration in Huh7 cells (n = 6 independent experiments). o Measurement of ChREBP transcriptional activity on ChoRE-luc reporter construct (n = 6 independent experiments). p Representative clonogenic assays shown (n = 6 independent experiments). All error bars represent mean ± SEM. All statistical analyses were made using two-way ANOVA and Tukey’s multiple-comparisons test. Source data are provided as a Source Data file.