Fumarate colorimetric assay kit

Levels of glucose, lactate, citrate, fumarate and ATP were measured using Glucose Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA), Lactate Colorimetric/Fluorometric Assay Kit (BioVision), Citrate Colorimetric/Fluorometric Assay Kit, Fumarate Colorimetric Assay Kit (BioVision) and ATP Colorimetric/Fluorometric Assay Kit (BioVision) respectively following the methods provided by the manufacturer. Briefly, conditioned mediums and cell lysates were moved into upper filter devices of Amicon Ultra-0.5 10KDa spin filters (Millipore, MA, USA) and centrifuged for 20 min at 8,000g to be deproteinized, then the solutions in the lower centrifuge tubes were used as the samples for analysis immediately. The fresh samples filtrated from conditioned mediums were used for glucose and lactate assay, and samples from cell lysates were used for intracellular citrate, fumarate and ATP measurement by corresponding kits. The amounts of glucose uptake and lactate secretion were calculated using the concentration difference value between conditioned mediums and fresh mediums. Metabolites levels were normalized by total protein concentration of cells.

We measured fumarate in frozen mouse fecal samples using the Fumarate Colorimetric Assay Kit (Biovision, Milpitas CA) following the manufacturer’s instructions. Data points below the lower detection limit of 200 nmol were not shown on plots, but were included in post-hoc statistical analyses as a pseudo-count of 200 nmol when all measures for a treatment group were below the detection limit. These measurements were collected for only a single cohort of mice due to availability of samples.

Nuclei were isolated from cells with nuclear expression of IDH-2 and PDHC or cells treated with DRN as per the experimental requirements. As per the requirement of the protocol, the samples were prepared using assay buffer or were deproteinated using 13% TCA and precipitated at 15,000×g for 5 min. The pH of the supernatant was adjusted with 2 N KOH to 7.5–8. The concentration of alpha-ketoglutarate was measured using an Alpha Ketoglutarate (alpha KG) Assay Kit (Abcam, ab83431). Acetyl-CoA was measured using a PicoProbe™Acetyl-CoA Fluorometric Assay Kit (BioVision, K317). Pyruvate, malate, succinate, and fumarate were measured using a Pyruvate Assay Kit (Sigma, MAK332-1KT), Malate Colorimetric Assay Kit (BioVision, K637), Succinate Assay Kit (Colorimetric) (Abcam, ab204718), and Fumarate Colorimetric Assay Kit (BioVision, K633), respectively.