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

Figure 3. Disrupted mitochondrial thiol redox homeostasis phenocopies TCA cycle inhibition.

(A) Schematic diagram highlighting mito 1-Chloro-2,4-dinitrobenzene (mitoCDNB) reaction with glutathione and the formation of mito 1-S-glutathionyl-2,4-dinitrobenzene (MitoGSDNB) and intensity levels in DMSO-treated and mitoCDNB (10 μM)-treated Fh1fl/fl cells. (B) Volcano plot of glutathione-related metabolites and amino acids in mitoCDNB-treated Fh1fl/fl versus DMSO-treated Fh1fl/fl cells. p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger and Yekutieli - FDR = 5%. (C) U-13C-glutamine tracing (m + 10, m + 5, m + 4 and m + 3 labeling intensity and total isotopologue fraction distribution) in DMSO and mitoCDNB-treated Fh1fl/fl cells. (A–C) (24 hr timepoint) (n = 5–10 independent biological replicates). (A and C) Data are mean ± SEM. p Value determined by unpaired two tailed t-test or ordinary one-way ANOVA corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 3.

Figure 3—figure supplement 1. Glutamine fate tracing upon mitochondrial GSH depletion.

Figure 3—figure supplement 1.

(A) Interleaved box and whiskers plot of fold change in GSH and GSSG with FHIN-1 (20 μM)-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). p values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***. (B) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (total isotopologue fraction distribution) in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (C) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (m + 5 and m + 3 labeling intensity) in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (D) ATP/ADP, lactate/pyruvate and NADH/NAD+ ratio in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). Data are mean ± SEM. p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.