Figure 3.

(a) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. (b) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 10⁶) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. (c) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μg/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. (d) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μg/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1^R140-expressing cells transfected; R172, IDH1^R172-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001