Fig 2: Oligodendroglioma cells produce 2HG in culture.Concentrations of 2HG in the medium from spheres and non-dissociated tissue from the xenograft relative to spheres from a freshly dissociated GBM without IDH mutation were measured over time. Measurements were made in triplicate. The decrease in 2HG concentrations is concomitant with the removal of approximately 50% of the spheres for implantation in between the 21- and 28-day time points.

Fig 3: IDH1 R132 H mutation status is not associated with a detectable difference in 5hmC content.Tumors were evaluated for immunoreactivity with antibodies specific for the IDH1 mutant R132H and the corresponding 5hmC levels were correlated with IDH1 immunopositive and immunonegative (IDH1 negative) tumors in (A) grade II astrocytoma, (B) combined low grade gliomas (C) anaplastic astrocytomas, and (D) glioblastoma. No significant difference was identified in 5hmC staining level within any of the specific tumor types.

Fig 4: Dimerization schematics for wild type and mutant IDH1 and IDH2 homo- and heterodimers conjugated to their respective Venus constructs(a) Schematic to demonstrate the principles of BiFC when the N- and C-termini of Venus protein are fused to the N-termini of proteins A and B, respectively. Protein-Protein interaction permits formation of a Venus-based bimolecular fluorescence complex resulting in fluorescence. Image modified from [17]. (b) Schematics to show IDH1 wild type and mutant homo- and heterodimers. (c) Schematics to show IDH2 wild type and mutant homo- and heterodimers. Dimerization between IDH monomers permits Venus complex formation via association of the N-terminal (VN) and C-terminal (VC) subunits leading to fluorescence. PTS1; peroxisomal targeting sequence type 1, MTS; mitochondrial targeting sequence, VN; N-terminal Venus (amino acids 1-173), VC; C-terminal Venus (amino acids 155-238). The Tobacco Etch Virus (TEV) linker region is located between IDH and the Venus construct. ★ indicates the R132H and R172K mutations for IDH1 and IDH2, respectively.

Fig 5: Validation of a newly developed TERT-specific antibody (TMab-6) usable for human glioma tissue. (a) Endogenous TERT was immunoprecipitated with an anti-human TERT mAb (TMab-6) followed by an RNA-dependent RNA polymerase (RdRP) assay using HeLa cells treated with nocodazole. Arrows indicate RdRP products. The preimmune (without antibodies) and irrelevant IgM lanes are a negative control, and the 10E9-2 antibody (MBL, Nagoya, Japan) lane is a positive control. nt, nucleotide. (b) The IDH-mutant astrocytoma (anaplastic astrocytoma: AA) and IDH-wildtype diffuse astrocytoma (Histologically anaplastic oligoastrocytoma: AOA) cases were analyzed for TERT protein expression. TMab-6 specifically recognized the nuclei of the tumor cells, and immunoreactivity was not detected in the negative control section without the application of TMab-6. An antibody absorption test with synthetic neutralization peptide of TERT further validated the specificity of TMab-6 as an anti-TERT antibody which is applicable to human glioma tissue. (c) TMab-6 Immunostaining of U87 malignant glioma cell lines with a TERT hotspot mutation (C228T) showed strong nuclear immunoreactivity for TERT. Ab, antibody; mt, mutation. Scale bar = 40 µm.