Fig 1: Identification of IDH1R314C in a high grade astrocytoma.(A) Sequencing trace showing the heterozygous C to T mutation at position 940 in the IDH1 gene found in the E98 cell line and (B) primary tumor material. This mutation leads to the p.Arg314Cys substitution in the IDH1 protein. (C) Display of the 3D- structure of human IDH1R314C generated with YASARA (PDB-file 3inm). The IDH1WT homodimer is displayed with bound cofactor NADP+ (yellow), and the arginine residue on position 314 (red), both subunits are coloured in a different shade of grey. The inset shows the mutated cysteine (red) superimposed on the original arginine (green).
Fig 2: IDH1R314C-BAPHIS expression in glioma cell lines leads to reduced forward activity compared to IDHWT expression.(A) Western blots showing expression of IDH1-BAPHIS in transiently transfected HEK293t and lentivirally transduced LN229 and U251 glioma cells. Blots were stained with anti-IDH1PAN and anti-IDH1R132H as indicated, or with anti-GAPDH as a loading control. Since the recombinant proteins are ~3 kDa larger than endogenous IDH1 they can be distinguished from the endogenous IDH1. Control LN229 and U251 were transduced with empty vector virus (EV). (B) Monitoring of NADPH formation after isocitrate/NADP+ addition to extracts from transfected/transduced cell lines. NADPH formation was monitored by absorbance measurements at 340 nm. Note that IDH1R314C-BAPHIS was far less effective in oxidative carboxylation of isocitrate than IDH1WT-BAPHIS. (C) Km values of IDH1-GST for NADP+. The Km value for NADP+ for IDH1R132H-GST could not be determined. (D) D-2-HG production by IDH1-BAPHIS expressing cell lines was measured with LC-MS. Only IDH1R132H-BAPHIS expressing cells were capable of D-2-HG production.
Fig 3: Survival curves of marker proteins.The relationship of individual proteins evaluated by immunohistochemistry with survival with different cut-off points. A. 14-3-3ß (positive v negative immunoreactivity), B. PHB (positive v negative immunoreactivity), C. IDH1 (negative/weak immunoreactivity v moderate/strong immunoreactivity), D. LDHB (negative/weak immunoreactivity v moderate/strong immunoreactivity), E. TCTP (negative/weak immunoreactivity v moderate/strong immunoreactivity), F. IDH1 (negative/weak/moderate immunoreactivity v strong immunoreactivity), G. MVP (negative/weak/moderate immunoreactivity v strong immunoreactivity), H. survival in each of 10 clusters identified by hierarchical cluster analysis (each cluster is numerically identified and corresponds to the clusters that are identified in the cluster analysis panel of Figure 7), I. survival in 2 clusters- cluster 1 and clusters 2–10 combined and J. two protein signature of 14-3-3ß and ALDH1 showing that double negative tumours have a significantly better outcome.
Fig 4: Homodimeric IDH1R314C-GST is defective in the isocitrate-to α-KG reaction and does not produce D-2-HG.(A) SDS-page gel stained with coomassie brilliant blue showing expression of purified IDH1-GST constructs. (B) Fluorescent monitoring of NADPH formation at 340 nm shows that only purified IDH1WT-GST is capable to convert isocitrate to α-KG under the reaction conditions tested, whereas IDH1R132H-GST and IDH1R314C-GST are inactive. (C) α-KG production by IDH1-GST enzymes was measured with LC-MS. Note that only IDH1WT-GST is capable of isocitrate-to-α-KG conversion. (n.d. = non detectable, area < 150). (D) Fluorescent monitoring of NADPH reduction shows that only purified IDH1R132H-GST is active in the α-KG-to-D-2-HG conversion. Both IDH1WT-GST and IDH1R314C-GST lack the capability to produce D-2-HG.
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