Figure 4

Mutant IDH1 activates glycolysis in IBOs through the upregulation of Pfkp. (A) The level of Pfkp expression in the indicated IBOs by RT-qPCR (n = 4, *P < 0.05, NS not significant). (B) Protein expression levels of Pfkp, Pfkm, and Pfkl as determined by western blotting. (C,D) Enzymatic assay of PFK-1 activity in the indicated IBOs. Time course of optical absorbance at 450 nm in (C), and comparison of PFK-1 activity in (D) (n = 4, *P < 0.05, **P < 0.01). (E) Gene expression of Pfkp in mut-IBOs transfected with shRNA for Pfkp (shPfkp-2, 5) or scrambled shRNA (Scr) as control (n = 4, *P < 0.05). (F) Organoid-forming efficiency of the indicated IBOs (>100 μm) at 7 days after plating (n = 4, 3000 cells per group, *P < 0.05). (G) Gene expression of Pfkp in mut-IBOs treated with 20 μM AGI-5198 (+) or DMSO vehicle (−) (n = 4, *P < 0.05). (H) IBOs established from wild-type mice were treated with 10 mM 2-HG (+) or vehicle (−) upon serial passage. Gene expression of Pfkp at passage 4 by RT-qPCR (n = 3, P = 0.14). (I) The level of trimethylation of histone H3 lysine4 (H3K4me3) on the promoter region of Pfkp by ChIP analysis in IBOs. The upper panel shows the schematic diagram of the transcriptional start site (TSS) and amplicons (0.2 kb and 50 kb downstream of the TSS). The lower graph shows ChIP- qPCR data (relative to input DNA, n = 4, *P < 0.05).