DHG and LHG inhibition experiments were performed in the presence of mmolL isocitrate or mmolL aKG and mmolL DHG or mmolL LHG or solvent only in the enzyme activity reaction medium.The HG:isocitrate and HG:aKG ratios used in these experiments are in line with the pathophysiological conditions in human glioma where DHG concentrations may be up to to, fold higher than isocitrate and aKG concentrations. Five to cellscm were seeded; higher densities are needed at higher IR doses to obtain sufcient amounts of colonies.A, the NADP dependent IDH activity of earlypassage IDHWTWT and IDHWTRH HCT cells at various isocitrate concentrations was determined as absorbance of blue formazan produced from NBT per cell.B, U and LN glioblastoma cell lines were stably transduced with lentiviral vectors harboring IDHWT and IDHRH genes.The lower band is endogenous IDH; the upper band is the tagged IDH.AntipanIDH antibody was used to detect IDHWT and IDHRH.Dq values were found by solving the semilog line equation for. Cells were permeabilized with pTNBS for hour and stained forgHAX foci using a mouse monoclonal antig HAX antibody for min at room temperature.Lastly, cover slips were washed with sTNBS, and nuclei were stained with DAPI for minutes at room temperature.Photomicrographs were obtained using custommade software. Stack images of at least cells per sample were taken.One stack consisted of slices with a nm interval between the slices along the zaxis.The images were processed using deconvolution Flunisolide software, and the number ofgHAX foci per cell was automatically scored using custommade software.The vulnerability of lDHWTRH and lDHWTWT HCT cells to IR was investigated using colonyforming assays.To test this, we treated lDHWTRH and lDHWTWT HCT cells with the NADPH surrogate and ROS scavenger NAC at hours before IR.The clonogenic fraction is the number of colonies counted divided by the number of cells plated, corrected for the plating efciency.We conrmed the increased radiosensitivity of lDHWTRH HCT cells in DMEM medium, which contains no reduced glutathione. CIMP has profound effects on gene expression and, theoretically, this could alter IR sensitivity.We compared genomewide methylation levels in earlypassage lDHWTRH and lDHWTWT HCT cells and observed a relative CIMP in P compared with P lDHWTRH HCT cells.Longterm culture did not induce CIMP in lDHWTWT HCT cells did not differ from IR sensitivity of P lDHWTRH HCT cells, and IR sensitivity is thus not related to CIMP.Of note, the radiosensitizing effect of DHG was larger in lDHWTWT than in lDHWTRH HCT cells, in line with preexisting endogenous DHG in lDHWTRH HCT cells.To study this, we performed enzyme activity experiments in the presence and absence of DHG or LHG.For both IDH and aKGDH, LHG was a more efcient IDH inhibitor than DHG.We validated these results in IDHRH U and LN cells. Larger amounts of IDHRH protein may require higher AGI doses for complete IDHRH inhibition.There was no effect after hour incubation with AGI on NADP dependent IDH activity in lDHWTWT HCT cells, in agreement with AGI specicity for IDHRH. Because AGI inhibits IDHRC as well, although at higher concentrations than IDHRH. Under steadystate conditions, lDHWTRH HCT cells had similar NADP NADPH ratios, GSHGSSG ratios, and ROS levels as lDHWTWT HCT cells, as determined by colorimetric and uorometric assays and uorescence ow cytometry experiments.

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