Fig. 4

Subcellular targeting of the D2HGlo sensor reveals that D-2-HG levels are elevated in the cytosol, nucleus and mitochondria of IDH1-R132H mutant U87MG glioma cells. (A) Left: Representative image of a wildtype IDH1 U87MG cell expressing Cyto-D2HGlo. Scale bar is 25 μm. Right: Comparison of the average FRET ratio in wildtype IDH1 and IDH1-R132H mutant U87MG cells expressing Cyto-D2HGlo. The cells were grown in the presence or absence of the IDH1-R132H inhibitor AG-120 (3 µM) for 48 h prior to collecting images. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 48 cells obtained from three independent experiments (AG-120-treated WT), n = 61 cells from four independent experiments (untreated R132H) and n = 47 cells from three independent experiments (AG-120-treated R132H). (B) Left: Representative image of a wildtype IDH1 U87MG cell expressing Nuc-D2HGlo. Scale bar is 25 μm. Right: Comparison of the average FRET ratio in wildtype IDH1 and IDH1-R132H mutant U87MG cells expressing Nuc-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 58 cells from four independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (C) Left: Representative image of a wildtype IDH1 U87MG cell expressing Mito-D2HGlo. Scale bar is 25 μm. Right: Comparison of the average FRET ratio in wildtype IDH1 and IDH1-R132H mutant U87MG cells expressing Mito-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 47 cells obtained from three independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 48 cells from three independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (A–D) Statistical analysis was performed using a one-way ANOVA test with post hoc Tukey (****, P < 0.0001 compared with untreated WT, AG-120-treated WT and AG-120-treated R132H; *, P < 0.05 compared with AG-120-treated WT and AG-120-treated R132H)