Growth arrest and cell survival of FL5.12 cells transfected with the TEF expression vector. (A) Growth curves of FL5.12 cells transfected with the empty vector, pMT-TEF, pMT-TEF/BX, or pMT–E2A-HLF. Cells were adjusted to 1 × 105 cells per mL and cultured in IL-3–containing medium in the presence (•) or absence (○) of 100 μM Zn. Representative data of multiple experiments using 3 independent pools are shown. Bars indicate standard error. (B) Growth of FL5.12 cells transfected with the empty vector or pMT-TEF in IL-3–containing medium in the presence of 0, 25, 50, 75, or 100 μM of Zn. (C) Growth curves of pMT-TEF–transfected FL5.12 cells after withdrawal of Zn. □ indicates the growth of cells cultured in IL-3–containing medium in the absence of Zn. Cells were preincubated in the presence of 100 μM Zn for 12 hours, washed with medium, adjusted to 1.5 × 105 cells per mL, and then cultured in IL-3–containing medium either in the presence (•) or absence (○) of 100 μM Zn. One hundred twenty hours after withdrawal of Zn, cells cultured in the absence of Zn were adjusted to 1 × 105 cells per mL and cultured in the presence (•- - -•) or absence (○- - -○) of 100 μM Zn. The means of triplicate samples are indicated. (D) Time course analysis of TEF expression in pMT-TEF–transfected FL5.12 cells after withdrawal of Zn. Cells were preincubated in the presence of 100 μM Zn for 12 hours, washed with medium (lane 1), then cultured in IL-3–containing medium in the presence (lanes 2, 4, and 6) or absence (lanes 3, 5, and 7) of 100 μM Zn for the indicated periods of time and subjected to immunoblot analysis using the HLF(C) antiserum. The expression of tubulin was also analyzed as control. (E) Cell cycle analysis of pMT-TEF–transfected FL5.12 cells. Cells cultured in IL-3–containing medium (top; - 12 hours) were preincubated with 100 μM of Zn for 12 hours (middle; 0 hour), washed with medium, and subsequently cultured in IL-3–containing medium in the absence of Zn for 72 hours (bottom; 72 hours). ▨ indicates G0/G1; ▪, S; and □, G2/M. (F) Number of viable cells after IL-3 deprivation of FL5.12 cells. FL5.12 cells transfected with the empty vector, pMT-TEF, pMT-TEF/BX, or pMT–E2A-HLF were precultured in IL-3–containing medium in the presence or absence of 100 μM of Zn for 16 hours. Cells were then washed with IL-3–free medium, adjusted to 5 × 105 cells per mL, and cultured without IL-3 either in the presence (and ▪) or absence (□) of Zn, respectively. Upon deprivation of IL-3 for 48 or 72 hours, the numbers of viable cells as determined by trypan-blue dye exclusion are indicated. The mean data of 3 independent experiments are shown. Bars indicate standard error.
