Figure 4
Figure 4. Enforced GATA-2 expression alters expression of genes involved in cell-cycle control but does not confer quiescence by MEF repression or CKI induction. (A) Quantitative RT-PCR of C/EBPα, Gfi-1, Egr2, Pu.1, and MEF in CB CD34+CD38− cells on enforced expression of GATA-2. Expression relative to HPRT (n ≥ 4; error bars indicate SEM). P < .01 for all. (B) Enforced expression of MEF from an LNGFR-expressing bicistronic construct does not prevent the conferral of quiescence by enforced expression of GATA-2 in CD34+ cells. Cells cotransduced with all combinations of GFP- and LNGFR-marked expression vector, empty or encoding GATA-2 or MEF, respectively, were stained with Hoechst and Pyronin Y. Enforced MEF expression has positive effects on cell cycle independently of GATA-2, but the effect of enforced GATA-2 expression is dominant to this. (C) CKIs p16INK4A, p19INK4D, p21CIP1, p27KIP1, and p57KIP2 are not induced by enforced GATA-2 expression in CB CD34+CD38− cells by quantitative RT-PCR relative to HPRT (n ≥ 4l error bars indicate SEM). (D) Enforced expression of GATA-2 in CB CD34+ cells does not cause induction of p27KIP1 as measured by intracellular flow cytometry. (E) Enforced expression of GATA-2 induces quiescence in p21−/− mouse Lin−Kit+Sca+ cells as measured by Hoechst/Pyronin staining, but (F) p27KIP1 is not induced in these cells. (G) Expression of CCND3, CDK4, CDK6, and MCM5 is reduced by enforced GATA-2 expression in CB CD34+CD38− cells, whereas HES1 expression is induced (quantitative RT-PCR relative to HPRT). (n ≥ 4 except n = 1 for HES1; P < .05 for all except MCM5 and HES1. MCM5 expression is reduced in 4 of 5 independent experiments and as a result P > .05). (H) Expression of CDK4, CDK6, and β-tubulin by Western blot in CD34+ cell extracts prepared from freshly isolated unstimulated cells and cells transduced with empty vector and GATA-2 after 3 days in SCF, TPO, and Flt3L. Densitometric analysis of expression relative to β-tubulin, normalized to the ratio in vector-transduced cells, is shown below each panel.

Enforced GATA-2 expression alters expression of genes involved in cell-cycle control but does not confer quiescence by MEF repression or CKI induction. (A) Quantitative RT-PCR of C/EBPα, Gfi-1, Egr2, Pu.1, and MEF in CB CD34+CD38− cells on enforced expression of GATA-2. Expression relative to HPRT (n ≥ 4; error bars indicate SEM). P < .01 for all. (B) Enforced expression of MEF from an LNGFR-expressing bicistronic construct does not prevent the conferral of quiescence by enforced expression of GATA-2 in CD34+ cells. Cells cotransduced with all combinations of GFP- and LNGFR-marked expression vector, empty or encoding GATA-2 or MEF, respectively, were stained with Hoechst and Pyronin Y. Enforced MEF expression has positive effects on cell cycle independently of GATA-2, but the effect of enforced GATA-2 expression is dominant to this. (C) CKIs p16INK4A, p19INK4D, p21CIP1, p27KIP1, and p57KIP2 are not induced by enforced GATA-2 expression in CB CD34+CD38− cells by quantitative RT-PCR relative to HPRT (n ≥ 4l error bars indicate SEM). (D) Enforced expression of GATA-2 in CB CD34+ cells does not cause induction of p27KIP1 as measured by intracellular flow cytometry. (E) Enforced expression of GATA-2 induces quiescence in p21−/− mouse Lin−Kit+Sca+ cells as measured by Hoechst/Pyronin staining, but (F) p27KIP1 is not induced in these cells. (G) Expression of CCND3, CDK4, CDK6, and MCM5 is reduced by enforced GATA-2 expression in CB CD34+CD38− cells, whereas HES1 expression is induced (quantitative RT-PCR relative to HPRT). (n ≥ 4 except n = 1 for HES1; P < .05 for all except MCM5 and HES1. MCM5 expression is reduced in 4 of 5 independent experiments and as a result P > .05). (H) Expression of CDK4, CDK6, and β-tubulin by Western blot in CD34+ cell extracts prepared from freshly isolated unstimulated cells and cells transduced with empty vector and GATA-2 after 3 days in SCF, TPO, and Flt3L. Densitometric analysis of expression relative to β-tubulin, normalized to the ratio in vector-transduced cells, is shown below each panel.

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