Ligand-activated GATA-2/ER chimeric form reversibly induced quiescence in a manner reproduced with constitutive lentiviral expression of FLAG–GATA-2. (A) β-Estradiol (1 μM) induces G₀/G₁ accumulation of Ba/F3 cells expressing GATA-2/ER (top) concomitant with increased quiescence as measured by DNA staining with Hoechst 33342 and polysomal RNA staining with Pyronin Y (bottom). One of 3 representative experiments is shown, with the percentage of G₀/G₁ cells falling in the G₀ gate indicated. (Bi) Withdrawal of ligand allows cells to progress into S phase and to exit quiescence, as assessed by reduced G₀/G₁ proportions indicated (top) and increased staining with Pyronin Y (bottom). (Bii) Proportion of G₀/G₁ cells falling in the G₀ gate from panel Bi are plotted. One of 2 representative experiments is shown. (C) Cell growth, attenuated by GATA-2/ER activation, resumes on ligand withdrawal. (D) Return to normal cell growth lags behind reduction in the proportion of quiescent cells as measured by Hoechst/Pyronin Y. (E) Bicistronic lentiviral expression construct used to drive enforced expression of FLAG-tagged GATA-2 and GFP. (F) Constitutive expression of FLAG–GATA-2 also increases quiescence of Ba/F3 cells growing in IL-3. The mean of 2 representative experiments is shown. Error bars indicate SEM. (G) The GATA-2–transduced cells are outgrown by untransduced cells in a GATA-2–dependent manner; n = 2 experiments.