Figure 3
Mutant IDH1 promotes cell-cycle transition independently of 2-hydroxyglutarate. (A) Representative FACS blots of cell-cycle phases in HoxA9-immortalized vector CTL, IDH1wt, or IDH1mut transduced (GFP+) bone marrow cells 9 weeks after transplantation (BrdU injections IP at 36, 24, and 12 hours before harvest). Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS. The upper quadrant represents cells in S phase; the lower left quadrant represents cells in G0/G1 phase; and the lower right quadrant represents cells in G2/M phase of the cell cycle. (B) Cell-cycle distribution in HoxA9-immortalized vector CTL, IDH1wt, and IDH1mut transduced (GFP+) bone marrow cells 9 weeks after transplantation (BrdU injections IP at 36, 24, and 12 hours before harvest). Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS (mean ± SEM). (C) Gene expression levels of cyclin-dependent kinase inhibitors in in vitro–cultured HoxA9 cells transduced with IDH1wt and IDH1mut. Gene expression was determined by RT-PCR relative to the housekeeping gene Abl and was normalized to gene expression in IDH1wt cells (mean ± SEM). (D) Methylation analysis of the CpG island in the Cdkn2b (p15) promoter in Hoxa9 cells transduced with IDH1wt and IDH1mut. DNA from in vitro–cultured cells was extracted and bisulfite treated. CpG methyltransferase-treated DNA from IDH1mut cells served as a positive control. The p15 promoter CpG was amplified and subcloned. Twenty clones were sequenced per cell type. Exons are represented by vertical rectangles. White horizontal bars above the exons show CpG islands; black horizontal bars below the exons show the tested region. An open circle indicates a nonmethylated CpG; a black circle indicates a methylated CpG. The percent methylation of the CpG island is calculated by the proportion of methylated CpGs from all investigated CpGs. (E) Methylation analysis of the CpG island in the Cdkn2a (p16) promoter in Hoxa9 cells transduced with IDH1wt and IDH1mut. The analysis was performed as described in the Figure 3D legend. *P < .05; **P < .01; ns, not significant.

Mutant IDH1 promotes cell-cycle transition independently of 2-hydroxyglutarate. (A) Representative FACS blots of cell-cycle phases in HoxA9-immortalized vector CTL, IDH1wt, or IDH1mut transduced (GFP+) bone marrow cells 9 weeks after transplantation (BrdU injections IP at 36, 24, and 12 hours before harvest). Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS. The upper quadrant represents cells in S phase; the lower left quadrant represents cells in G0/G1 phase; and the lower right quadrant represents cells in G2/M phase of the cell cycle. (B) Cell-cycle distribution in HoxA9-immortalized vector CTL, IDH1wt, and IDH1mut transduced (GFP+) bone marrow cells 9 weeks after transplantation (BrdU injections IP at 36, 24, and 12 hours before harvest). Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS (mean ± SEM). (C) Gene expression levels of cyclin-dependent kinase inhibitors in in vitro–cultured HoxA9 cells transduced with IDH1wt and IDH1mut. Gene expression was determined by RT-PCR relative to the housekeeping gene Abl and was normalized to gene expression in IDH1wt cells (mean ± SEM). (D) Methylation analysis of the CpG island in the Cdkn2b (p15) promoter in Hoxa9 cells transduced with IDH1wt and IDH1mut. DNA from in vitro–cultured cells was extracted and bisulfite treated. CpG methyltransferase-treated DNA from IDH1mut cells served as a positive control. The p15 promoter CpG was amplified and subcloned. Twenty clones were sequenced per cell type. Exons are represented by vertical rectangles. White horizontal bars above the exons show CpG islands; black horizontal bars below the exons show the tested region. An open circle indicates a nonmethylated CpG; a black circle indicates a methylated CpG. The percent methylation of the CpG island is calculated by the proportion of methylated CpGs from all investigated CpGs. (E) Methylation analysis of the CpG island in the Cdkn2a (p16) promoter in Hoxa9 cells transduced with IDH1wt and IDH1mut. The analysis was performed as described in the Figure 3D legend. *P < .05; **P < .01; ns, not significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal