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. 2021 Dec 23;10:e72593. doi: 10.7554/eLife.72593

Figure 7. Atf4 regulates amino acid and GSH metabolism.

(A) Western blot analysis of Atf4 and β-actin as loading control (24 hr timepoint) (representative image of 3 independent biological replicates) and qPCR analysis of Atf4-target genes (24 hr timepoint) (n = 3 independent biological replicates) in DMSO-, FHIN-1- and AA5-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4. Data are mean ± SEM. p value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (B) Volcano plot of glutathione-related metabolites and amino acids in Atf4-silenced Fh1fl/fl cells versus non-targeting control (NC)- Fh1fl/fl cells (48 hr timepoint). (C) Heatmap of amino acid levels in FHIN-1- and AA5- versus DMSO-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint). (B–C) (n = 5 independent biological replicates). (D) Schematic diagram highlighting key findings of the comparative analysis between acute FHi versus SDHi.

Figure 7.

Figure 7—figure supplement 1. Changes in glutathione metabolism intermediates upon Atf4 silencing and acute TCA cycle inhibition.

Figure 7—figure supplement 1.

(A) Interleaved box and whiskers plot of fold change in glutathione-related metabolites in DMSO- versus FHIN-1 (20 μM)-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint) (n = 5 independent biological replicates). (B) Interleaved box and whiskers plot of fold change in glutathione-related metabolites in DMSO- versus AA5 (1 μM)-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint) (n = 5 independent biological replicates). (A–B) p value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger and Yekutieli - FDR = 5%. p < 0.05*; p < 0.01**; p < 0.001***.