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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

From: Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A

Fig. 3

Optimization of the culture protocol for efficient erythroid differentiation from human-derived erythroblastic K562 cells. A Schematic diagram describing the original (Protocol 0) and the modified protocols (Protocols 1−4) for the induction of erythroid differentiation using EPO-based medium in K562 cells. (B, C) Cell viability B and the percentages of CD71+ and CD235a+ cells C at various times of culture from different protocols. Data are mean ± SD (n = 3). *P < 0.05 versus Protocol 0 on the same day of culture; two-sided Student's t test. D Western blot analysis of cellular O-GlcNAcylation and its cycling enzymes, erythroid-associated proteins, and globin proteins along the course of differentiation (day −1 to day 10). Blots were reprobed with anti-β-actin antibody to establish a loading control. E Quantitative analysis of O-GlcNAc, OGA, OGT (upper, left), erythroid-associated proteins (upper, right), and globin proteins (lower) by densitometry after normalization to the loading control is shown. Data are mean ± SD (n = 3). *P < 0.05 versus cells at the start of culture (day −1); two-sided Student's t test. F qPCR of genes encoding key proteins in D. Data were normalized to GAPDH and represented in a heatmap

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