EHMT1 or EHMT2 knockdown increases γ-globin gene expression and HbF synthesis in primary adult human erythroid cells. (A) Validation of shRNA-mediated knockdown of EHMT1 or EHMT2 in primary adult human erythroid cells by qRT-PCR at day 14 of erythroid differentiation (mean ± SD, n = 5-6 biological replicates). (B) Validation of shRNA-mediated knockdown of EHMT1 or EHMT2 by western blot analysis in primary adult human erythroid cells at day 9 of erythroid differentiation and after 4 days of puromycin selection. shRNA-mediated knockdown of EHMT1 or EHMT2 increases γ-globin mRNA levels (mean ± SD, n = 5-6 biological replicates) (C) and HbF levels assessed by HPLC in primary adult human erythroid cells at day 14 of erythroid differentiation (mean ± SD, n = 4-6 biological replicates) (D). (E) Representative HPLC chromatograms showing HbF abundance. (F) 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 = 3-5 biological replicates). **P < .01; ***P < .001.