(a) Western blot analysis of IDH1 and IDH2 in subcellular fractions of D2HGDH models (knockdown, left panel; ectopic expression, right panel) shows modulation of IDH2 levels. Densitometric quantification is shown at the bottom. (b) Top—western blot analysis of IDH2 KD cells expressing an empty-MSCV vector or WT D2HGDH. Middle—Expression of WT D2HGDH decreased H3K methylation (compare lanes marked with a red star); IDH2 KD restored the H3K methylation levels in these cells. Bottom—expression of WT D2HGDH increased HIF1α hydroxylation and decreased total HIF1α levels (compare lanes marked with a red star); IDH2 KD reversed the increase in HIF1α hydroxylation (and decrease in total HIF1α) associated with expression of D2HGDH WT. (c) Cells expressing D2HGDH-WT (and an empty pLKO.1) displayed a significantly higher abundance of 5hmC marks (top) and a concomitant decrease in global DNA methylation (botton) than MSCV-pLKO.1 controls (marked by red star; P<0.001 two-tailed, Student’s t-test). IDH2 KD significantly lowered or increased the levels of 5hmC and 5mC marks, respectively, in D2HGDH-WT cells (P<0.0001, ANOVA, P<0.001 Bonferroni’s multiple comparison test), with a more modest change in MSCV-expressing controls. These data are mean and s.d. of an assay performed in triplicate, which was confirmed in an independent biological replicate. The data from the WBs were confirmed in two to three biological replicates. (d) Top—western blot analysis of IDH2 ectopic expression in D2HGDH KD cells. Middle—D2HGDH KD elevated H3K methylation (compare the three first lanes) and this change was abrogated by expression of IDH2. Bottom—D2HGDH KD decreased HIF1α hydroxylation and increased its total levels (compare the three first lanes); these changes were absent in D2HGDH KD cells ectopically expressing IDH2. (e) D2HGDH KD decreased the abundance of 5hmC marks (top) and increased global DNA methylation 5mC (bottom; <0.0001, ANOVA); expression of IDH2 restored these values to that of control (pSIL-MSCV) isogenic cells. These data are mean and s.d. of an assay performed in triplicate, confirmed in a biological replicate. The western blot data shown in d were confirmed in biological replicates.