Fig. 6. Augmented EryP generation and cell proliferation by GATA2. (A) GATA2 induction scheme in differentiating EBs and formation of EryP colonies. Above is the scheme used to generate Blast colonies. Uppermost numbers indicate EB day. Lower gray bars indicate duration of Dox treatment and subsequent treatment of replated EBs. (B) EryP potential of D4 iGATA2 EBs treated with or without Dox at D2 or D3. Values are mean±s.e.m., from four independent experiments; ^*, P<0.05 versus untreated. (C) Representative bright field images (100x) of EryP colonies from D4 EBs replated in the absence (left) or presence (right) of Dox. (D) BrdU incorporation in EryP cells treated with or without Dox during replating. Values are normalized by subtracting background absorbance and then the BrdU incorporation in untreated EryP cells is normalized to 1. Values are mean±s.e.m. from six independent experiments; ^*, P<0.005 versus untreated. (E) Cell cycle gene expression profile of iGATA2 EryPs with or without Dox during EryP colony formation. EryP cells from day 2 or 3 of EryP colony formation were harvested and RNA utilized for qRT-PCR assays. Genes were normalized against Gapdh and then the ratio of +Dox to -Dox determined, where values <1 were assigned their inverse to give a negative fold change. Values are mean±s.e.m. from four independent RNA sets.