Extended Data Fig. 4: Glutarate modulates mitochondrial function by inhibiting pyruvate dehydrogenase.
From: Glutarate regulates T cell metabolism and anti-tumour immunity
a, PDHc activity of CD8+ T cells transduced with a NTC or shGCDH normalised to total protein. Two-tailed unpaired t-test; n = 3. b-c, Quantitative analysis of MST and GST following acute (30 min) (left) and chronic (7 days) (right) DEG exposure, as described in Fig. 4c-d and Fig. 4h-i, shown as log2 FC relative to untreated control (black dotted line). Two-tailed one sample t-test; Acute GST n = 6; Acute MST n = 6; Chronic GST n = 5, Chronic MST n = 10. d, Protein expression of PDHK1 as determined by western blot analysis. Ordinary one-way ANOVA; n = 7. e, Protein expression of phosphorylated PDHE1α (ser239) as determined by western blot analysis. Ordinary one-way ANOVA; n = 7. f, Protein expression of PDHc as determined by western blot analysis. Ordinary one-way ANOVA; n = 8. g, Glucose uptake by human CD8+ T cells treated with DEG. Data represented as relative light units (RLU). Mixed-effects analysis; n = 9 (acute), n = 14 (chronic). h, qPCR analysis of CD8+ T cells treated with DEG for 7 days. Each dot represents one human donor relative to untreated donor control (dotted black line), normalised to HPRT. Two-tailed one sample t-test; FASN n = 4, FASBP n = 4; CPT1A n = 3. i, Model of mitochondrial fuel flexibility tests and illustration of calculations used to determine mitochondrial fuel oxidative capacity and dependency. (Adapted from BioRender.com) j, Fatty acid oxidation dependency as determined by OCR measurements during a mitochondrial fuel flex test of CD8+ T cells treated with +/- DEG 500 µM for 7 days as described in Extended Data Fig. 4i. Calculated percentage fatty acid dependency (right): Two-tailed paired t-test; n = 5. k, OCR measurements and percentage fuel oxidative capacity (cap) or dependency (dep) during a mitochondrial fuel flex test of CD8+ T cells treated with +/- DEG 500 µM for 7 days, as described in Extended Data Fig. 4i. Calculated percentage fuel capacity and dependency (bottom): Two-tailed paired t-test; Glucose oxidation dependency n = 6; Glucose oxidation capacity n = 5; Glutamine oxidation dependency n = 5; Glutamine oxidation capacity n = 6. All experiment describes used 500 µM DEG. All scatter plots show median and 95% CI where each dot represents one human donor. OCR time course graphs show Mean and Error with 95% CI.
