FIG 9.
SREBP promotes 2-hydroxyglutarate production in IDH1R132C mutant cells. (A) Cells were lysed for immunoblotting at 72 h posttransfection with nontargeting siRNAs (−) or siRNAs targeting SREBP1 and SREBP2 or IDH1. (B) Diagram of carbon flux from [U-13C]glucose into 13C-2-HG (M+2). Filled and empty circles represent 13C and 12C carbons, respectively, with M+6 and M+2 referring to the mass increase from the stable isotope carbons. (C to E) Normalized peak areas of 13C-labeled (M+2) and unlabeled (M+0) metabolites and the fractional abundance of 13C-labeled M+2 metabolites measured by LC-MS/MS in metabolite extracts from cells at 72 h posttransfection with nontargeting control siRNAs (−) or siRNAs targeting SREBP1 and SREBP2 or IDH1. HT1080 cells (C) and isogenic HCT116 cells with wild-type (+/+) or mutant (R132C/+) IDH1 (D) were labeled with [U-13C]glucose for 20 min. For panel E, HT1080 cells were labeled with [U-13C]glucose for 24 h, and the results of two experiments are shown. In panels C and D, the data are representative of at least two independent experiments. In panels C, D, and E, the data are presented as means ± the SEM of biological triplicates (*, P < 0.05 relative to cells transfected with control siRNAs; #, P < 0.05 relative to wild-type HCT116 cells).