UNC0638 increases human γ-globin and HbF expression and mouse embryonic β-globin gene expression in a dose-dependent manner. (A) Fold change in HBA1/2, HBB, and HBG1/2 mRNA levels relative to GAPDH in primary adult human erythroid cells at day 14 of erythroid differentiation and after 10 days of UNC0638 treatment (mean ± standard deviation [SD], n = 5-6 biological replicates). (B) Relative expression of γ-globin genes in primary adult human erythroid cells at day 14 of erythroid differentiation and after 10 days of UNC0638 treatment (mean ± SD, n = 5-6 biological replicates). (C) HbF levels assessed by HPLC in primary adult human erythroid cells at day 14 of erythroid differentiation (mean ± SD, n = 4-8 biological replicates). (D) Representative HPLC chromatograms showing HbF abundance. (E) Flow cytometry analysis for F-cells: representative histograms showing the percentage of adult human erythroid cells expressing HbF and the fluorescence intensity relative to the cell number at day 14 of erythroid differentiation (mean ± SD, n = 3-4 biological replicates). ***P < .001. (F) Relative expression levels of the mouse embryonic globin genes Hbb-εy and Hbb-βh1 in MEL cells after 48 hours of UNC0638 treatment and in Ehmt2 knockout MEL cells (mean ± SD, n = 3 biological replicates). *P < .05. Cl., clone.