Characterization of LHGFR in HEK293FT cells

To characterize the sensitivity and specificity of LHGFR expressed in HEK293FT cells, cells were trypsinized 48 h following transfection and suspended in 1× Hank’s balanced salt solution supplemented with 20 mM HEPES. Increasing concentrations of l-2-HG or other compounds including glutarate, d-2-HG, and glucose was mixed with the cell suspensions in a 96-well plate, respectively. Digitonin at a concentration of 10 μM was used to induce cell permeabilization and deplete intracellular l-2-HG for in vivo response curves construction. Then, the fluorescence intensities were determined by a SpectraMax i3 fluorescence plate reader (Molecular Devices, USA) with excitation at 430 nm and emission at 485 nm (mTFP) and 528 nm (Venus). Basal l-2-HG concentration in HEK293FT cells under physiological conditions was determined by substituting the emission ratios of non-permeabilized HEK293FT cells into the calibrated in vivo response curves.

For the detection of hypoxia-induced production of l-2-HG, LHGFR_0N3C or LHGFR_3N7C was expressed in HEK293FT cells and cultured sequentially under normoxic conditions for 24 h and hypoxic conditions for 24 h in the absence or presence of 5 mM dimethyl-2-ketoglutarate (DMαKG). The preparation of cell suspensions and the measurement of emission ratios were performed using the same procedure. The background fluorescence was subtracted at each emission wavelength.