Requirement for ZBP-89 in human globin gene activation and erythroid maturation. (A) Lentiviral-mediated shRNA knockdown of ZBP-89 in ex vivo differentiated erythroid precursors. Western blot for ZBP-89 protein levels in nontransduced erythroid precursors (EryP), or those transduced with the empty lentiviral vector or vectors containing one of 2 shRNA constructs (sh1 and sh2) that target different sequences within ZBP-89 exon 9 (“shRNA knockdown”). Western blot was performed from cells harvested on day 7 after placement into differentiation medium. (B) May-Grunwald-Giemsa stains of cytospun EryP cells transduced with the empty vector, sh1 or sh2 on days 7 and 10 of differentiation (original magnification × 600; see supplemental Figure 2 for additional images). (C) Flow cytometric analysis for CD71 and CD235a expression of EryP cells transduced with the empty vector, sh1, or sh2 on days 7 and 10 of differentiation. (D) Gene Set Enrichment Analysis of genes differentially expressed on ZBP-89 knockdown (sh1; vs the empty vector) on day 6 of differentiation compared with the erythroid-specific expressed gene set.23 The y-axis shows the enrichment score for each gene, which is illustrated as a vertical line plotted in rank order of most up regulated (left) to most down-regulated (right). (E-F) Quantitative RT-PCR analysis of mRNA transcript levels for the indicated genes in EryP cells transduced with the empty vector, sh1, or sh2 on day 7 (E) and day 10 (F) of differentiation. Expression is shown relative to levels from the empty vector-transduced cells and is normalized to Gapdh levels. Measurements represent the mean from > 3 independent experiments ± SEM. *Significant differences compared with the empty vector (P < .05, Student t test).
