Ectopic expression of cyclin D1 and Cdk4 results in an increase in polyploidization and cell size of GATA-1 KD megakaryocytes. (A-D) Ploidy analysis of BSA gradient–purified GATA-1 KD megakaryocytes was performed using flow cytometry of DAPI-stained cells. Cells were doubly infected with the MigR1 containing EGFP and pCS containing hCD25. Cyclin D1 was cloned into MigR1 and Cdk4 was cloned into pCS allowing for double infection and expression of cyclin D1 alone, Cdk4 alone, or cyclin D1 and Cdk4. Double-infected cells were selected through gating of EGFP-positive and CD25 double-positive cells. Bars indicate the percentage of cells that have DNA content of 8N or greater. One representative of 3 experiments is shown. (E-H) Cell size analysis performed using flow cytometry of the infected cells described in panels A-D. Double-positive cells were analyzed using histograms of forward scatter–area to compare cell size. Values for the mean FSC-A are indicated for each population of doubly infected cells. One representative of 3 experiments is shown.