Figure 3. Expression of 2HG-producing IDH proteins increases global 5-methylcytosine levels.

(A) 293T cells were transiently transfected with empty vector, wild-type or R132H mutant IDH1, or wild-type or R172K mutant IDH2. After 3 days, cells were lysed and assessed for IDH1 expression levels by Western blot, and then re-probed for IDH2. β-actin antibody was used as a control. (B) Cells transfected in parallel to those lysed in (A) were extracted for intracellular metabolites. Metabolites were then derivatized with MTBSTFA and analyzed by GC-MS. Shown is the quantitation of 2HG signal intensities relative to the intrasample glutamate signals for a representative experiment. (C) Global DNA methylation levels in cells were analyzed 3 days following transfection by immunofluorescence using antibody against 5-methylcytosine. Quantification of fluorescence intensities from one experiment is shown. Data is representative of three independent experiments. (D) 32D cells were transduced with empty retroviral vector or with wild-type or R172K mutant IDH2, selected in 2.5 µg/ml puromycin for 7 days, and then lysed to confirm stable expression of IDH2. Tubulin antibody was used as a control. (E) Cells were extracted for their intracellular metabolites which were then derivatized with MTBSTFA and analyzed by GC-MS. Shown are representative gas chromatographs from wild-type and mutant IDH2 expressing cells depicting the derivatized metabolites eluting between 31.3 and 33.5 min, including 4-oxoproline (4-oxo Pro), glutamate (Glu), and 2HG. Metabolite abundance refers to GC-MS signal intensity. (F) DNA was extracted from cells with stable wild-type or mutant IDH2 expression, and global DNA methylation levels were measured by slot blot using antibody against 5-methylcytosine. Relative intensity of signals of three independent experiments was quantified. Error bars: +/− SD for triplicate experiments. (See also Figure S4)