Effect of TET3 knockdown on human erythroid differentiation. (A) Expression of TET3 as assessed by quantitative RT-PCR, with β-actin as internal calibrator. Error bars indicate SEM (n = 3). (B) Flow cytometric analysis of erythroid progenitor populations. Cells cultured for 6 days were stained with antibodies against IL-3R, GPA, CD34, and CD36. The IL-3R and GPA double-negative population was further separated by CD34 and CD36. The IL-3R⁻GPA⁻CD34⁺CD36⁻ population was identified as BFU-E and the IL-3R⁻GPA⁻CD34⁻CD36⁺ population was identified as CFU-E. The data are shown as mean ± SEM of 3 independent biological replicates. (C) The colony forming ability of sorted progenitor cells was assessed by colony forming assay. Error bars indicate SEM (n = 3). (D) The expression of GPA on day 7 of culture: the GPA⁺ cells were gated and the percentages of GPA⁺ cells are shown as mean ± SEM of 3 independent biological replicates. **P < .01. (E) Terminal erythroid differentiation was monitored on indicated days by flow cytometric analysis based on the expression of band 3 and α4 integrin. Representative plots of α4-integrin vs band 3 of GPA⁺ cells are shown and the erythroblasts are separated into 5 populations: proerythroblasts (I), early basophilic erythroblasts (II), late basophilic erythroblasts (III), polychromatic erythroblasts (IV), and orthochromatic erythroblasts (V). mRNA, messenger RNA; SEM, standard error of the mean.