Figure 3.

Succinate oxidation in mitochondria. (A) Citrate synthase activity (nmol * min⁻¹ * mg⁻¹ protein) with or without prior incubation with 5 mM succinate for 6 h (N ≥ 3). (B) Dehydrogenases activity measured after incubation with 5 mM succinate for 6 h using a WST-1 assay (N ≥ 11). (C) Time-course of ROUTINE respiration of living cells after incubation with 5 mM succinate for 1, 3 or 6 h (N ≥ 4). Measurements were performed with living cells in their respective medium without supplements. (D) O₂ consumption stimulated by addition of succinate (10 mM) after rotenone titration (N ≥ 12). Representative traces show the titration steps of the SUIT protocol: ce1, living cells (ROUTINE respiration); ce2Rot, rotenone (0.5 µM Rot for inhibition of CI, measurement of residual O₂ consumption); ce3S, succinate (10 mM S; respiration supported by external succinate plasma membrane transport in living cells); ce4D, ADP (1 mM ADP) and ce4c, cytochrome c (10 µM c for testing the cell viability of living cells and plasma membrane integrity); 1Dig, digitonin (5 µg/mL Dig; plasma membrane permeabilization enabling the entrance of substrates for stimulation in O₂ consumption - OXPHOS); and 2Ama, antimycin A (2.5 µM Ama for inhibition of CIII). All experiments were performed at 37 °C with a cell concentration of 1.0 × 10⁶ x/mL suspended in their respective cell culture media without supplements in the 2 mL O2k-chamber. Units are O₂ flow per cell counts, I_O2 [amol*s⁻¹*x⁻¹], where “x” represents the elementary unit of cell count [29], baseline corrected (bc). (E) ATP content after incubation of cells for 1 or 6 h with (+) or without (−) 5 mM succinate, 0.5 µM rotenone, 5 mM malonate (N ≥ 3). Median and IQR. Unpaired t-tests for independent experiments comparing +/− succinate in each condition (A–D) or ordinary one-way ANOVA tests (E). * p = 0.05; ** p < 0.001; **** p < 0.00001.