Figure 5.

IDH1 Arg-132 mutant down-regulation of p53 depends on HIF-2α. A, IDH1 Arg-132 mutant increases HIF protein levels in transgenic mice livers. HIF protein levels in mice livers of the indicated genotypes were determined by Western blotting. B, knockdown of HIF-1α has no effect on p53 down-regulation caused by IDH1 Arg-132 mutant. IDH1^WT/WT and IDH1^WT/Mut MEFs were infected with lentiviruses expressing HIF-1α shRNA or control shRNA. 48 h after infection, cells were treated with or without 2.5 μm DOX, followed by Western blotting to detect the indicated proteins (left). Quantitation of signal intensities of Western blot (WB) bands of p53 (right) was performed by using ImageJ software. p53 levels were normalized to β-actin levels. Data are presented as the mean ± S.D. of three independent experiments (N.S. = not significant). C, IDH1 mutant down-regulation of p53 depends on HIF-2α. IDH1^WT/WT and IDH1^WT/Mut MEFs were infected with lentiviruses expressing HIF-2α shRNA or control shRNA. 48 h after infection, cells were treated with or without 2.5 μm DOX, followed by Western blot to detect the indicated proteins (left). Quantitation of signal intensities of Western blot bands was performed and presented as in A (right). D, HIF-1β knockdown reverses p53 suppression by the IDH1 Arg-132 mutant. IDH1^WT/WT and IDH1^Mut/Mut MEF were infected with lentiviruses expressing HIF-1β shRNA or control shRNA. 48 h after infection, cells were treated with or without 2.5 μm DOX, followed by detection of the proteins as indicated (left). Quantitation of signal intensities of Western blot bands was performed and presented as in A (**, p < 0.01; ***, p < 0.001, unpaired Student's t test).