FIGURE 3.
Cellular 2HG accumulation from cytosolic IDH1 mutation depends on maintained expression of wild-type IDH1. A, depiction of the open reading frames of the homologous proteins cytosolic IDH1 and mitochondrial IDH2. Recurrently mutated amino acid residues are marked with an asterisk (*). Arg-132 of cytosolic IDH1 is structurally analogous to Arg-172 of mitochondrial IDH2. B, FLAG-tagged IDH2 R140Q, IDH2 R172K, and IDH1 R132H (at various doses), or empty vector were transfected into 293T cells. Forty-eight hours post-transfection, cells were harvested and assessed for 2HG accumulation by GC-MS (top) or protein expression by Western blot (bottom). Data are representative of mean ± S.D. of 3 biological replicates from 2 independent experiments. C, a similar transfection as in B was performed with or without the co-transfection of Myc-tagged wild-type IDH1. D, Myc-tagged IDH1 R132H engineered to be siRNA-resistant, with or without FLAG-tagged IDH1 WT also engineered to be siRNA resistant, or empty vector, were transfected into 293T cells along with siCTRL or siRNA targeting only endogenous wild-type IDH1 (siIDH1). siIDH1 transfection to knockdown endogenous IDH1 levels was reproducibly associated with greater expression levels of Myc-tagged mutant IDH1, but this was still accompanied by a significant decrease in 2HG accumulation unless FLAG-tagged wild-type IDH1 was co-transfected. Data are representative of mean ± S.D. of 3 biological replicates from 4 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.