Fig. 4.

Maleate treatment and succinomimetic mutation of DLST reduce the activity of the KGDHC.A-C, KGDHC activity (A, n = 4), protein succination (B), and ATP production rate linked to glycolysis and mitochondrial function (C) in DLST KO H838 cells expressing WT hDLST, after treatment for 16 h with 0–5 mM maleate (n = 12). D, KGDHC activity in DLST KO H838 cells expressing WT hDLST or hDLST forms where Cys37 or Cys178 were mutated to Glu to mimic succination (n = 4). E, Respiratory profile of the cells described in (D). The oxygen consumption rate (OCR) was measured using a Seahorse XF analyzer, in basal conditions and after sequential injection of oligomycin (O), FCCP (F), and rotenone/antimycin A as described in Methods. F–H, Basal respiration (F), oxygen consumption coupled to ATP production (G), and maximal respiration (H), in the experiment described in (E). I, Extracellular acidification rate (ECAR) after oligomycin injection in the experiment described in (E), n = 6/group.In panels A, C, D, and F–I, data expressed as mean ± SEM and comparisons by one-way ANOVA and post hoc Student-Neuman-Keuls test (A and C, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for 1 mM and 5 mM maleate vs. 0 mM maleate, and ^##p < 0.01 for 5 mM maleate vs. 1 mM maleate; D, and F–I, **p < 0.01, and ****p < 0.0001 for mutants vs. WT, and ^#p < 0.05 and ^###p < 0.001 for C178E vs. C37E). In panel B, the molecular weight markers are shown on the left side.