Effects of GATA-2 on the growth of mIL-3–dependent cell lines, Ba/F3, 32D, and FDC-P1.

(A) NIH3T3 cells were transfected with 2 μg of a reporter gene for GATA, 3 × MαP-Luc, 10 ng pRL-CMV-Rluc, and 2 μg of an expression vector of GATA-2/ER or an empty expression vector. After 12 hours, the cells were serum deprived and cultured for 24 hours. Then, the cells were stimulated with 1 μM estradiol for 8 hours and lysed in the lysis buffer, followed by the measurement of the firefly and therenilla luciferase activities. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the renilla luciferase activities. The results are shown as the means ± SD of triplicate experiments. (B) The expression levels of ectopic (Ect) GATA-2/ER and endogenous (End) GATA-2 were examined in the transfectants by immunoblot analysis. (C) The cells of Ba/F3, 32D, and FDC-P1 each transfected with an expression vector of GATA-2/ER were seeded at a cell density of 100/μL and cultured with (▪) or without (■) 1 μM estradiol. Total number of viable cells was counted by trypan blue dye exclusion method at the times indicated. The results are shown as means ± SD of triplicate cultures. (D) Cells cultured as described above were subjected to PI staining, and DNA content was analyzed on FACSort. Cell cycle analysis was performed with a program Modfit LT2.0. The results shown are representative of 3 independent experiments.