Fig 2: Immunoreactivities of the proteins indicated on the left in brain tissue homogenates (indicated on the top; individual samples coded by a three-digit number obtained from brain regions as detailed in Table 3). The most-left lane includes purified, recombinant proteins (Prot.) for SUCLG1, SUCLG2, OGDH, DLST, and DLD

Fig 3: 28-day tail suspension promotes the expression of ACO2, DLST and CS in hippocampus mitochondria of rats.(A) Protein levels of ACO2, DLST and CS were determined by immunoblotting. (B) The relative expressions of ACO2, DLST and CS were represented by the intensity ratio between the protein and the loading control (VDAC) in each lane. n = 4, error bars indicated standard deviations. *p<0.05; **p<0.01 compared with each control group.

Fig 4: Mitochondrial membrane potential (MMP) and α-ketoglutarate dehydrogenase (KGDH) activities are associated with ferroptosis. (A, B) MMP and lipid ROS changes in HN9 cells stably transduced with shDLD, DLDres, or a vector control (vtr). The cells were subjected to CC deprivation or 1 mM SAS at different time points, and then, MMP and lipid ROS were incubated with tetramethylrhodamine ethyl ester (TMRE, 200 nM) and BODIPY, respectively, 30 min before each indicated time point. **P < 0.001 relative to a vector control or shDLD plus DLDres. (C, D) Quantification of KGDH activity and succinate in HN9 cells stably transduced with shDLD, DLDres, or vtr. The cells were also exposed to CC deprivation or 1 mM SAS for 4 or 8 h *P < 0.01 relative to control or between different groups with or without shDLD or DLDres. (E–G) Cell death assay in HN3 and HN9 cells with or without dihydrolipoyl succinyltransferase (DLST) gene silencing. The cells were transfected with siDLST or scrambled siRNA (siCtr) and then subjected to CC deprivation. Western blots were performed in the cells with siDLST or siCtr. Scale bar, 50 μm. NS indicates statistically not significant.

Fig 5: Impairing mitochondrial function altered succinylation and protein distribution in the whole cell as well as in the mitochondria and non-mitochondrial fractions.a The effects of rotenone (100 nM/20 min) on succinylation in HEK cells. After separation, mitochondrial and non-mitochondrial fractions were immune-precipitated with anti-succinyl lysine antibody and separated by SDS-PAGE followed by western blotting. The data from three different replicate experiments were expressed as the mean with error bars from standard error of the mean (SEM) (n = 3 independent experiments, two-way ANOVA followed by Bonferroni’s multiple comparisons test). b The effects of rotenone (100 nM or 5 μM/20 min) on the distribution of KGDHC protein between mitochondria and non-mitochondrial fractions. The data from three different replicate experiments were expressed as the mean with error bars from SEM (n = 3 independent experiments, two-way ANOVA followed by Tukey’s multiple comparisons test). c The effects of rotenone (100 nM, 5 μM/20 min) on the distribution of PDHC protein between mitochondria and non-mitochondrial fractions. The data from three different replicate experiments were expressed as the mean with error bars from SEM (n = 3 independent experiments, two-way ANOVA followed by Tukey’s multiple comparisons test). d Rotenone induces release of DLST into cytoplasm. In the control conditions, DLST (magenta) was concentrated inside mitochondria defined by COX-IV labeling (green). After 1 h of 100 nM Rotenone treatment, additional DLST labeling was found in the cytoplasm. Inserts on the right are magnified regions. Magenta: DLST; Green: COX-IV; Error bars represent SEM deviation from the mean (n = 98 fields from 19 dishes, two-way ANOVA followed by Bonferroni’s multiple comparisons test). Source data are provided as a Source Data file.