Fig. 2. ABHD11 is required for OGDHc function.
a Schematic of the TCA cycle (oxidative metabolism) and reductive carboxylation (reductive metabolism), illustrating the fate of 13C carbons upon incubation with [U-13C5]-glutamine. b–g Stable isotope tracing of control HeLa cells compared to mixed CRISPR KO populations (sgRNA) of ABHD11 or OGDH incubated with [U-13C5] glutamine. 2-oxoglutarate (b), succinate (c), fumarate (d), malate (e) and citrate (f), divided by metabolite isotopologues (m + 0 to m + 5) are indicated. Two biologically independent replicates, n = 5 technical replicates per sample, mean ± SD (g) OGDHc activity in isolated mitochondria. Mitochondria were extracted from control or mixed CRISPR KO populations of ABHD11, OGDH or VHL HeLa cells and OGDHc activity measured by a redox sensitive colorimetric probe for 2-OG oxidation. n = 3 biologically independent samples, mean ± SEM, **p = 0.0082, two-tailed t test. h Bioenergetic assays of oxygen consumption rates (OCR) in control, ABHD11 deficient or OGDH deficient HeLa cells (mixed KO populations). ABHD11 and OGDH were depleted as described, and analysed by using a Seahorse XFe24 Extracellular Flux Analyzer (n = 4 technical replicates per sample, mean ± SD). Three basal measurements were made at 9 min intervals followed by three measurements per treatment (1 μM oligomycin, 1 μM FCCP and 1 μM antimycin/rotenone). OCR was normalised to total cell number. i Comparison technical repeats at first basal measurement from (h); ***p = 6.3 × 10−5, two-tailed t test. j Measurement of 2-hydroxyglutarate (2-HG) levels following [U-13C5] glutamine stable isotope tracing in control HeLa cells compared to mixed CRISPR KO populations (sgRNA) of ABHD11 or OGDH. Metabolite isotopologues (m + 0 to m + 5) are indicated. Two biologically independent samples are shown; n = 5 technical replicates per sample, mean ± SD. k Relative quantification of 2-HG enantiomers upon derivatisation with diacetyl-L-tartaric anhydride and LC-MS analysis. l, m Inhibition of lactate dehydrogenase A (LDHA) in ABHD11 deficient HeLa cells. Mixed CRISPR KO ABHD11, VHL or PHD2 cells were treated with sodium oxamate (Ox) (l) or GSK-2837808A (m) as indicated for 24 h. HIF-1α levels were measured by immunoblot.