FIG. 7.
Overexpression of ROD1 inhibits TPA-induced megakaryocytic differentiation of human K562 cells. The ROD1 transfectants and empty vector transfectants were cultured in growth medium containing 10 nM TPA. On the day indicated, cells were stained with anti-CD61–fluorescein isothiocyanate (FITC) monoclonal antibody followed by FACScan analysis. (A) Time course of the emergence of CD61-positive cells during the induction of megakaryocytic differentiation. The percent population of CD61-positive cells was calculated by dividing the number of FITC-positive cells by the number of total cells. The mean percents CD61-positive cells in ROD1 overexpressors (Rod1-C2, -C3, -C7, -C9, and -C10) or Neo clones were plotted on a graph with the standard deviation. The right figures show the FACS patterns of the Neo2 and Rod1-C2 after TPA treatment for various times. (B) The ROD1 overexpressors (Rod1-C2 and Rod1-C9) and negative controls (Neo2 and Neo4) were induced for differentiation by culture in medium containing 0.6 nM TPA for 3 days, then stained with anti-CD61–FITC monoclonal antibody and analyzed by using an argon laser microscope (Zeiss). (C) Rod1 overexpression blocks the induction of the PDGF gene, another early differentiation marker. The human Rod1 transfectants and negative controls were cultured in medium containing 10 nM TPA and harvested at the indicated times. Cell lysates (32 μg of protein each) were then separated by SDS–15% PAGE, and the PDGF-B was detected by Western blotting with an anti-PDGF-B polyclonal antibody. The arrowhead indicates the PDGF-B protein migrating as a 30-kDa band upon SDS-PAGE. (D) ROD1 overexpression blocks the induction of CD49b, a late marker of megakaryocytic differentiation. The human ROD1 overexpressors (Rod1-C2 and Rod1-C9) and the negative controls (Neo2 and Neo4) were cultured in medium containing 50 nM TPA. After a 3-day incubation, the cells were stained with anti-CD49b–FITC monoclonal antibody and analyzed by using an argon laser microscope (Zeiss).