The enforced expression of wt HCP in SKT6 cells inhibits EPO-induced globin expression, and hemoglobinization. (A through C) SKT6 cells were electrotransfected with pCINeowtHCP or pCINeo expression vectors, and stably transfected lines were isolated by selection in G418 (see the Materials and Methods). In assays of EPO-induced hemoglobinization and globin expression, cell lines were cultured at 2.5 × 105 cells/mL in the presence or absence of EPO (±2 U/mL). At 24, 48, and 72 hours of EPO exposure, frequencies of hemoglobinized cells were assayed by staining with DAF. Values are the means ± standard deviations of three replicate cultures. At each time interval, lysates also were prepared for assays of globin expression levels via Western blotting. For each lane, lysate from 2 × 105 cells was analyzed. For SKT6-wtHCP cells, data are for cell lines prepared from two independent transfections.
(D) In assays of EPO-induced Stat5a activation, SKT6 cell lines (parental SKT6.1B cells, and stably transfected SKT6.1B-wtHCP cells) were exposed to EPO (±50 U/mL) for 15 minutes. Lysates then were prepared and were incubated with a biotinylated PRE cassette. Levels of activated Stat5a were assayed by adsorption of Stat5a-DNA complexes to streptavidin agarose CL4B and ECL Western blotting after the elution of bound factor from washed gels (upper gels). As a control, levels of Stat5a in total lysates also were assayed by Western blotting (lower panel).
