Succinate assay kit

Extracts and assay were performed using a commercial succinate assay kit (Abcam, Cambridge, UK), with minor modifications. The confluent wells of 6-well plates were rinsed in ice-cold PBS and resuspended in 100 μL of ice-cold SDH Assay Buffer (provided), both pre-equilibrated to the adequate O₂ levels (21%, 10%, 5% or 1% O₂). Cells were scrapped from wells using cell lifters, then transferred to pre-cooled microfuge tubes and homogenized by pipetting. Clear extracts were used for assay, and the conversion of substrate (DCIP, provided) was assessed by following changes in absorbance at 600 nm over time. Measurements in kinetic mode were taken continuously for 30 min, and a linear range was selected to calculate the changes of absorbance over time (DOD/DT). DOD was converted to nmol of DCIP following the subtraction of background and against a standard curve. Results were normalized to amount of protein used per well, quantified by BCA.

HT29 cells supernatant was used to quantify the succinate levels using the Succinate Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, supernatants were filtered with 10 kDa spin columns (ab93349, Abcam). The Reaction Mix from the kit was used to incubate samples in 96-well plates at 37 °C during 30 min. Finally, the absorbance at 450 nm was measured with the microplate reader SpectraMax Plus 384 (Molecular Devices, San Jose, CA, USA) and the succinate concentration was calculated using the standard curve.

Mitochondria were isolated by using the Mitochondria Isolation kit (#89874, Thermo, MA, USA) according to the manufacturer’s instructions. For hexokinase activity detection, mitochondria were isolated from cells and pellet was lysed and subjected to Hexokinase activity measurement by using Hexokinase Colorimetric assay kit (#K789-100, Biovision, CA, USA). G6PD activity measurement by using G6PD activity assay kit (#MAK015, Sigma-Aldrich, MO, USA). NADPH level performed using NADPH quantification kit (#V9510, Promega, WI, USA). NAD⁺/NADPH level were performed using NADP/NADPH quantification kit (#G9081, Promega, WI, USA). Sccinate levels were measured using a Succinate Assay Kit (#b204718, Abcam) and lactate Colorimetric/Fluorometric Assay kit (#K607-100, Biovision) according to the manufacturer’s protocol.