FIG. 5.

The Fli-1-GATA-1 combination results in synergistic activation of the megakaryocyte-specific promoters GPIX and GPIbα in HeLa cells. (A) HeLa cells were transfected with 200 ng of the GPIX-567 luciferase reporter plasmid alone (bar 1) or together with increasing amounts of expression plasmids for Fli-1 (bars 2 and 3, respectively, 200 and 400 ng) or were transfected with 200 ng of GATA-1 expression plasmid together with increasing amounts of Fli-1 (bars 4 to 6, respectively, 100, 200, and 400 ng) or increasing amounts of GATA-1 expression plasmid alone (bars 7 to 9, respectively, 200, 400, and 800 ng). Cells were harvested 48 h posttransfection in passive lysis buffer, and 10 μl was used in the luciferase assay. Values are expressed as mean increases ± standard deviations relative to a value of 1 for each reporter. Results shown are means from three experiments performed in triplicate. (B) Western blotting was performed on 30 μg of total cell extract from a single representative experiment described in panel A and probed for Fli-1 or GATA-1 expression to rule out possible effects of GATA-1 expression on Fli-1 levels and vice versa. Fli-1 and GATA-1 are indicated by the arrows. (C) HeLa cells were transfected with 800 ng of the GPIbα-253 luciferase reporter plasmid alone (bar 1) or together with increasing amounts of expression plasmids for Fli-1 (bars 2 and 3, respectively, 100 and 200 ng); 50 ng of GATA-1 expression plasmid together with increasing amounts of Fli-1 (bars 4 to 6, respectively, 50, 100, and 200 ng); or increasing amounts of GATA-1 expression plasmid alone (bars 7 to 9, respectively, 10, 20, and 50 ng). Cells were harvested and assayed as described for panel A. (D) Western blotting, as described for panel B, on 50-μg total cell extracts from a single representative experiment from panel C.