Fig. 2: Liver tissue from Gclc KO mice has induced NRF2 target genes and repressed lipogenic gene expression.

A GSH quantity (normalized to mg tissue weight) in the liver, kidney, lung, and brain from Gclc WT mice (n = 4). A one-way ANOVA with subsequent Dunnett’s multiple comparisons test was used to determine statistical significance (Liver vs. Kidney P value < 0.0001, Liver vs. Lung P value < 0.0001, Liver vs. Brain P value < 0.0001). B Representative H&E-stained liver slides in WT and KO mice. Scale bars = 500 µm. Data shown is representative of at least three replicates. C Relative abundance of GSH precursors and their related metabolites in the serum of WT (n = 4) and KO (n = 4) mice. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. KO: Cysteine P value = 0.9987, Glutamate P value = 0.0278, Glycine P value = 0.9993, Hypotaurine P value = 0.0004, Glutamine P value > 0.9999, Serine P value = 0.0105). D Gene Set Enrichment Analysis (GSEA) of oxidative stress-related pathways in the liver of KO (n = 4) compared to WT (n = 4) mice. Enrichment p-values were calculated using an adaptive multi-level split Monte Carlo scheme and were corrected for multiple testing using Benjamini and Hochberg false discovery rate. E Proteomic analysis of upregulated liver proteins in KO (n = 6) compared to WT (n = 6) mice. Black data points = proteins with P value < 0.05 and log2 fold change >1. Red data points = annotated NRF2 target proteins. F Relative mRNA expression of annotated NRF2 target genes in the liver of WT (n = 4) and KO (n = 4) mice. Expression levels were normalized to the expression of the reference gene Rps9. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. KO: Nqo1 P value < 0.0001, Hmox1 P value < 0.0001, Txnrd1 P value = 0.2255, Gclm P value = 0.7934). G Representative immunoblot analysis of NQO1 in the liver of WT and KO mice. Data shown is representative of at least three replicates. H Representative immunofluorescence images of the liver from WT and KO mice stained with an antibody against NQO1. Scale bars = 200 µm. Data shown is representative of at least three replicates. I Schematic of the proposed mechanism of NRF2-dependent repression of lipogenic gene expression. J GSEA of lipogenic-related pathways in the liver of KO (n = 4) compared to WT (n = 4) mice. Enrichment p values were calculated using an adaptive multi-level split Monte Carlo scheme and were corrected for multiple testing using Benjamini and Hochberg false discovery rate. K Proteomic analysis of downregulated liver proteins in KO (n = 6) compared to WT (n = 6) mice. Black data points = proteins with P value < 0.05 and log2 fold change > 1. Red data points = annotated lipogenic proteins. L Relative mRNA expression of annotated lipogenic genes in the liver of WT (n = 4) and KO (n = 4) mice. Expression levels were normalized to the expression of the reference gene Rps9. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. KO: Scd1 P value = 0.0160, Fasn P value = 0.0336, Acaa1b P value = 0.0162, Fabp1 P value = 0.0051). Indicated n values represent biologically independent samples from mice. Data are shown as mean ± SEM. ns = not significant, *P value < 0.05, **P value < 0.01, ****P value < 0.0001. (I) created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.