Fumarase

Freshly isolated cells were suspended in 0.5 ml of buffer containing 1 mM Tris–HCL (pH 7.4). Aconitase and fumarase activity were measured according to manufacturer’s instructions (Abcam). Aconitase measures the conversion of citrate to isocitrate, whereas fumarase measures the conversion of fumarate to malate. Enzymatic activity was quantified based on absorbance at 450 nm at 37 °C and results were normalized to protein concentration.

Activity assays for pyruvate dehydrogenase (PDH; Abcam, Cambridge, UK), α-ketoglutarate dehydrogenase (α-KGDH; Antibodies Online, Aachen, Germany), isocitrate dehydrogenase (IDH; Sigma-Aldrich), and fumarase (Abcam) were performed according to the manufacturer's instructions.

ADSCs were lysed using RIPA lysis buffer, and the total protein concentration was measured with a BCA protein assay kit (all from ThermoFisher Scientific). The transferred protein on the PVDF membrane (Millipore) was blocked with 5% non-fat milk for 1 h, followed by incubation with primary antibodies overnight at 4 °C. After washing with TBST, the PVDF membrane was incubated with the corresponding HRP-conjugated second antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. The primary antibodies used were: β-Actin (1:1,000), Tom20 (1:1000), Hexokinase 1 (HK1, 1:1000); Hexokinase 2 (HK2, 1:1000); Phosphofructokinase (PFK, 1:1000); Lactate dehydrogenase A (LDHA, 1:1000); Citrate Synthase (CS, 1:1000); Dihydrolipoamide succinyltransferase (DLST, 1:1000), Succinate dehydrogenase A (SDHA, 1:1000), and Fumarase (FH, 1:1000) (all obtained from Abcam). Mitochondrial fission factor (MFF, 1:1000); Mitofusin 2 (MFN2, 1:1000); Opticatrophy protein 1 (OPA1, 1:1000) (all obtained from Proteintech).