Reexpression of p18/p27 rescues cell cycle defect and enucleation in Eklf^−/− ex vivo cultures. (A) qRT-PCR of cell cycle genes before (day 0) and after (day 2) erythroid differentiation of Eklf^+/+ and Eklf^−/− ESRE cultures (n = 3). p18, a previously characterized EKLF target, and p27, a novel EKLF target, were significantly downregulated at day 2 in Eklf^−/− cells compared with Eklf^+/+ cells. (B) EKLF chromatin immunoprecipitation performed before (day 0) and after (day 2) erythroid differentiation of Eklf^+/+ ESRE culture (technical replicate; n = 3) shows that EKLF binds to Cdkn1b (p27) gene specifically during terminal differentiation, indicating that Cdkn1b is a novel direct target of EKLF. (C) Eklf^−/− ESREs were retrovirally transduced with Empty vector, EKLF wild-type, or CDKN2C (p18) constructs as indicated. Cells were stained with DRAQ5, and cell cycle flow cytometric analysis was performed at day 3 of erythroid differentiation. Cell cycle defects are rescued in CDKN2C (p18) transduced cells (n = 3). (D) Eklf^−/− ESREs were retrovirally transduced with Empty vector, EKLF wild-type, or CDKN1B (p27) construct as indicated. Cells were stained with DRAQ5, and cell cycle flow cytometric analysis was performed at day 3 of erythroid differentiation. Cell cycle defects are rescued in CDKN1B (p27) transduced cells (n = 3). (E) Quantification of enucleation percentage from biological replicates (n = 3) reveals robust rescue of enucleation in CDKN2C (p18) transduced cells. (F) Quantification of enucleation, gated among FSC low cells, at day 3 of differentiation from biological replicates (n = 3) demonstrates a partial rescue of enucleation in CDKN1B (p27) transduced cells. (G) MGG staining of enucleated cells that were sorted for Eklf^+/+, p18, or p27 transduced Eklf^−/− ESRE cultures at day 3 of differentiation. Scale bars correspond to 50 µm length. Original magnification ×40. Data for p18 are a composite image. *P < .05, **P < .01, ***P < .001.