Fig. 7

BCL11A is a key mediator of O-GlcNAc-mediated erythroid differentiation in K562 cells. A (left) DNA sequencing spanning over the Cas9 cut site on BCL11A from the CRISPR control (pLenti) cells in comparison with the double knockdown BCL11Ai/OGAi cells. The guide sequence is underlined in black, and the PAM sequence is denoted by a dotted red underline in the control sample. Vertical dotted lines denote the expected cut site. (right) Western blot analysis of BCL11A and OGA levels showing knockdown efficiency in BCL11Ai/OGAi cells in comparison with the single knockdown OGAi cells. (lower) The inferred sequences of BCL11A present in BCL11Ai/OGAi cells and their relative proportions (%INDEL) were listed along with the translated amino acids. B Percentages of CD71⁺ and CD235a⁺ cells at various times of culture in the optimized EPO-based medium. Data are mean ± SD (n = 3). ^*P < 0.05 versus pLenti cells on the same day of culture; one-way ANOVA with Tukey’s posttest. ^#P < 0.05 versus OGAi cells on the same day of culture; one-way ANOVA with Tukey’s posttest. C Representative morphology of differentiated cells on Wright-stained cytospin slides before imatinib pre-exposure (day −1) and after 10 days of culture (left) and its scoring for percentages of cells in different stages (right). Scale bar = 30 μm. D Western blot analysis of cellular O-GlcNAc, OGA, and OGT levels upon terminal erythroid differentiation at various times (day −1 to day 10). Blots were reprobed with anti-β-actin antibody to establish a loading control. The dashed lines separate juxtapose lanes taken from the same experiment and the same blot image. E Quantitative analysis of O-GlcNAc, OGA, and OGT levels by densitometry after normalization to the loading control is shown. Data are mean ± SD (n = 3). ^*P < 0.05 versus pLenti cells on the same day of culture; one-way ANOVA with Tukey’s posttest