Fig. 7

Effect of α-KG on c-Myc expression in AML cell lines and primary leukemia cells. a Western blotting analysis of IDH2 and C-MYC protein expression in U937 and ML-1 cells treated with the indicated concentrations of DM-αKG for 6, 12 and 24 h. b Relative protein levels of C-MYC in AML cell lines (data from three separate experiments) and primary leukemia cells from AML patients with wild-type IDH2 (n = 4) treated in vitro with the indicated concentrations of DM-αKG for 12 h. *p < 0.05, **p < 0.01, ***p < 0.001. c Relative mRNA level of C-MYC in U937 and ML-1 cells treated with the indicated concentrations of DM-αKG for 6 h. RNA expression was measured by RT-qPCR. ***p < 0.001. d Effect of DM-αKG (4 mM) on c-Myc mRNA stability. Cells were pre-treated with DM-αKG for 4.5 h followed by incubation with 5 μg/mL actinomycin D for the indicated time periods (0, 15, 30, 45, 60 and 90 min). The mRNA degradation rate was estimated according to a published method [52]. e C-MYC protein expression in U937 and ML-1 cells cultured with or without glutamine (Gln) for 24–48 h as indicated. f Western blotting of IDH2 and C-MYC protein expression in primary leukemia cells isolated from 6 AML patients with wild-type IDH2 treated ex-vivo with the indicated concentrations of DM-αKG for 6, 12 and 24 h. g Western blotting analysis of IDH2 and C-MYC protein expression in human primary AML cells from two patients with mutant IDH2 treated ex-vivo with indicated concentrations of DM-αKG for 6, 12 and 24 h