Fig 2: Inhibition of MDH-2 affects cell metabolism and fate. The accumulated during anoxia electron donors result in ROS overproduction during the reoxygenation and eventually in cell death through apoptosis and ferroptosis or cell senescence. Although by inhibiting the Krebs cycle, LW6 decreases electron donor production in the anoxia phase and protects the cells during reoxygenation, ATP is still produced, preventing cell energy collapse. In the latter, the pyruvate-malate cycle and the malate-aspartate shuttle may contribute, as well as the effect of LW6 on the transcription factors HIF-1α and p53. Both are downregulated by LW6 and affect the expression of critical enzymes of cell metabolism. In addition, downregulation of p53 by LW6 protects the cells from apoptotic cell death or senescence, while downregulation of ROS prevents ferroptotic cell death. FADH2, reduced flavin adenine dinucleotide; GLS-2, glutaminase-2; GLUT, glucose transporter; HIF-1α, hypoxia-inducible factor-1α; HK-II, hexokinase-II, LDH, lactate dehydrogenase; MDH-2, malate dehydrogenase; PDH, pyruvate dehydrogenase; NADH, reduced nicotinamide adenine dinucleotide; ROS, reactive oxygen species.

Fig 3: Normalized experimental and predicted enzymatic activity. The detection was based on ELISA assay and the activity was measured in mOD/min/mg of proteins. The data are normalized to the highest activity measured for these tests. Error bars of predicted fluxes are standard deviation of predicted values for each replicate. Dashed lines highlight the potential higher error rate for HP1 at day 9, as average viable cell density was lower than expected. CS, Citrate synthase; MDH, Malate dehydrogenase; PDH, Pyruvate dehydrogenase.

Fig 4: Anoxia-reoxygenation induces senescence phenotype, while LW6 prevents it. Anoxia-reoxygenation increases the p21 level, whereas the MDH-2 inhibitor LW6 prevents reoxygenation-induced p21 upregulation (A,B). Reoxygenation downregulated Ki-67, while LW6 prevented reoxygenation-induced Ki-67 reduction (A,C). Under reoxygenation, GLB-1 increased, but LW6 reversed the above change (A,D). RPTECs under reoxygenation produced more IL-1β, whereas LW6 downregulated reoxygenation-induced IL-1β overproduction (E). * p < 0.05 vs. control; # p < 0.05 vs. LW6-treated RPTECs under normoxia; + p < 0.05 vs. RPTECs under reoxygenation, ^ p < 0.05 vs. LW6-treated RPTECs under reoxygenation. GLB-1, beta-galactosidase; IL-1β, interleukin-1β.

Fig 5: MCJ silencing enhances β-oxidation and prevents lipid accumulation in hepatocytes in vitro and in vivo.a–j Mice were placed on mMCD diet 1 week prior to the initiation of the treatment and maintained on the diet for the duration of the study. Treatment with Invivofectamine-formulated siMCJ (n = 5) or siRNAc (n = 5) was given weekly for 3 weeks. Tissues were harvested 1 week after the last dose. a Oxygen consumption rate (OCR) using the Seahorse analyzer. b ATP levels in isolated mitochondria. c Rate of fatty acid oxidation determined by the production of acid soluble metabolites (ASM). d Rate of fatty acid oxidation determined by the production of CO2. e mRNA expression of fatty acid oxidation genes in siMCJ-treated liver relative to control liver, as determined by real time RT-PCR. f De novo lipogenesis of fatty acids (FA), diglycerides (DG), and triglycerides (TG) in the liver. g Triglycerides levels and (h) ketone bodies levels in serum. i Activity of malate dehydrogenase (MDH2) in liver extracts. j Glutathione (GSH) levels in liver. k-l Primary hepatocytes from WT mice were transfected with siRNAc or siMCJ and were incubated with steatosis-inducing doses of oleic acid alone for 24 h or in combination with rotenone for the last 6 h. k Representative images of lipid content and (l) quantification evaluated by Bodipy staining. *denotes p < 0.05, as determined by Student’s t test analysis. Error bars show standard error (SE) in all the panels except panels (e) and (f) where error bars show standard deviation (SD). Source data are provided as a Source Data file.