Fig 1: Folic acid and one-carbon donor availability regulate liver mitochondrial and lipid homeostasis. (A) Methylenetetrahydrofolate reductase (MTHFR) and (B) mitochondrially encoded cytochrome c oxidase II (MT–CO2) in livers of glycine N-methyltransferase (GNMT) knockout (KO) and wild-type (WT) mice fed a control (CON) or folate-supplemented (FS) as determined by immunoblotting with a (C) representative immunoblot (arbitrary units; n = 6–8 per group). (D) Liver succinate dehydrogenase subunit A (SDHA), (E) cytochrome c oxidase subunit 4 (COXIV), and (F) MT–CO2 in livers of WT mice receiving a high-fat diet (HF) and or a serine/glycine-deficient HF diet (-SG) as determined by immunoblotting with a (G) representative immunoblot (arbitrary units; n = 7–8 per group). (H) Liver phospholipids, (I) diacylglycerides, and (J) triacylglycerides (μg/mg; n = 7–8 per group). Data are presented as mean ± SEM. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001.
Fig 2: Mdivi-1 inhibits HCC tumor growth and metastasis in a xenograft murine model by restoring SDH and mito dynamics. A–D Huh7 cells were implanted subcutaneously into nude mice. Mice were treated intraperitoneally with vehicle and Mdivi-1 (25 mg/kg) twice weekly for three weeks. A Tumor volume was measured by calipers twice weekly. B Representative images of excised tumors harvested at the experimental endpoint and arranged in descending order of tumor size. C Tumor weight. Each dot denotes a mouse. D Lung metastatic area was quantified using ImageJ. E Plasma succinate concentration in mice before (baseline) and day 22 after Huh7 cell implantation with or without Mdivi-1 treatment. Succinate concentrations were measured using a Succinate Colorimetric Assay Kit. Data represent mean ± SEM (n = 5 for each group). F Protein levels of SDHA, SDHB, Opa1, Mfn1, and Mfn2 in excised tumors were assessed by immunoblotting with antibodies against the indicated proteins and β-actin. G Protein levels of p-Drp1 (Ser616) and Drp1 in excised tumors were assessed by immunoblotting. Densitometric quantifications for F and G are shown in the right panels. Data represent mean ± SEM of three mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001
Fig 3: Reduced SDHB and elevated succinate levels in HCC tissues and cells. A–D Human HCC tumor tissues. A SDHB mRNA levels in paired liver tumor specimens and adjacent non-tumor liver tissues (n = 150) were quantified by qPCR. B Kaplan–Meier plots of overall survival (n = 364) and relapse-free survival (n = 316) stratified by low versus high SDHB expression were generated using the TCGA liver cancer dataset and the Kaplan–Meier Plotter platform with the auto-selected best cutoff. C Serum succinate concentrations in healthy subjects (n = 47) and HCC patients (n = 150) were measured by UPLC coupled with a G6495C triple quadrupole mass spectrometer. D Kaplan–Meier plots of overall survival and relapse-free survival between low- and high-succinate levels in HCC patients stratified by low versus high serum succinate concentrations (n = 150). Data in A and C represent mean ± SEM. ****P < 0.0001. Data in B and D were analyzed by log-rank test. E–H SDH and succinate in HCC cells. E CM was collected after 48 h of incubation. Succinate concentrations in CM from THLE-2 vs Huh7 or HepG2 cells were assessed using a Succinate Colorimetric Assay Kit. F SDH activity in THLE-2 vs Huh7 or HepG2 cells was measured using a Succinate Dehydrogenase Activity Assay Kit. G Left panel: SDHA and SDHB proteins in Huh7 vs THLE-2 cells were analyzed by immunoblotting, and right panels: densitometric quantification. H SDHB mRNA levels were quantified by qPCR. Data in E–H represent mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001
Supplier Page from Abcam for Succinate Dehydrogenase Activity Assay Kit (Colorimetric)