Ikaros expression is regulated by the GATA switch during megakaryopoiesis. (A) Lineage-negative wild-type bone marrow cells were grown in SCF/THPO media for 4 days and RNA extraction was performed each day. Expression of Ikaros, Gata2, and Gata1 were assessed by qRT-PCR analysis. Data were normalized to day 1. Means ± SD are shown from 4 independent experiments. *P < .05; #P ≤ .01. (B) G1ME cells were transduced with retroviral vectors Migr1 or Migr1-Gata1, GFP sorted, and cultured for 2 days prior to RNA extraction. Data are shown as means ± SD from 3 independent experiments. *P ≤ .005. (C) Genome browser picture of Ikzf1 locus with the indicated GATA-2 (dark blue), GATA-1 (red), and switch (green) binding sites. Data were extracted from GATA-2 and GATA-1 ChIP-Seq experiments.8 (D) qRT-PCR performed on RNA extracted from day 2 GFP-sorted G1ME transduced with banshee, banshee-shGata2, Migr1, and Migr1-Gata1 retroviral vectors. Means ± SD of triplicate experiments are shown. *P ≤ .01. (E) Western blots performed with nuclear extracts from day 2 GFP-sorted G1ME cells transduced with banshee, banshee-shGata2, Migr1, and Migr1-Gata1 retroviral vectors. Quantification of band intensities relative to Hsc70 and normalized to empty vector Banshee or Migr are indicated.
