AGI-6780 releases TF-1 R140Q differentiation block by allowing EPO-induced demethylation; reduced histone mark in the γ-globin (HBG) promoter. (A) Pretreatment of TF-1 R140Q cells with AGI-6780 allows EPO-induced demethylation of the HBG promoter. (IDH2, EPO, and AGI-6780 conditions apply to all subfigures; error bars show standard deviation). (B) RT-qPCR for HBG expression shows that treatment of IDH2 R140Q cells with AGI-6780, which decreased DNA hypermethylation at this promoter, can restore expression of HBG. GAPDH expression used for control. (C) AGI-6780 induced dissociation of H3K9me3 from the HBG promoter (as measured by ChIP, using an anti-H3K9me3 antibody) and restored differentiation and expression of HBG, as measured by RT-PCR and the red color change of the cells in the microtubes. Fold enrichment of the HBG promoter with H3K9me3 compared with input DNA was determined by qPCR. Nonspecific enrichment was measured by immunoprecipitation with α-IgG and lack of association of H3K9me3, with the GAPDH gene promoter serving as controls. (D) Picture of the cell pellets and 2HG levels (mM) at cell harvest.