Figure 2. Increased metabolism in S47 cells compared to WT cells.

(A) Oxygen consumption rates (OCR) in WT and S47 LCLs were assessed using the Seahorse XF Mito Stress Test. OCR was measured first in basal conditions, and following injection of oligomycin, FCCP and rotenone/antimycin. The bar graph depicts maximal OCR after FCCP injection at ~40 min timepoint; data are representative of three independent experiments performed with at least six technical replicates, presented as mean ± SD. (B–C) Basal and compensatory glycolysis in WT and S47 LCLs (B) and MEFs (C) were assessed using the Seahorse Glycolytic Rate Assay. Basal glycolysis is first measured, followed by treatment of cells with rotenone/antimycin and 2-deoxy-D-glucose (2-DG). The bar graph depicts basal glycolysis at ~1 min timepoint and compensatory glycolysis after antimycin/rotenone injection at ~22 min timepoint; data are representative of three independent experiments performed with at least 10 technical replicates. Bar graphs are presented as mean ± SD. (D–E) Consumption of glucose and glutamine from media and production of lactate and glutamate were analyzed from homozygous WT, heterozygous WT/S47 and homozygous S47 human LCLs (D) and primary MEFs (E) using a YSI-7100 Bioanalyzer. Means and SEM are shown (n = 5).

Figure 2—figure supplement 1. Increased metabolism in S47 MEFs but no differences in mitochondrial content in WT and S47 cells.

(A–D) WT and S47 MEFs were incubated with 25 mM ¹³C-glucose for 15 min and the abundance of citrate (A), malate (B), aspartate (C), and glutamate (D) isotopologues was quantified by LC-MS/MS. Data are presented as mean ± SD, n = 3; two-way ANOVA. (*) p-value<0.05, (**) p-value<0.01, (***) p-value<0.001, (****) p-value<0.0001. (E) Mitochondrial mass in WT and S47 MEFs measured by Mitotracker Green fluorescence. Data depicted are representative of three independent experiments performed in triplicate. (F) Cell lysates extracted from WT, WT/S47 (het) and S47 LCLs and WT and S47 homozygous MEFs were subjected to western blot analysis and immunoblotted for TFAM, MTCO1, SDHA, TOMM20, and GAPDH (loading control).