Nrf2 activation reduces ROS levels and platelet gene expression. (A) Nuclear accumulation of Nrf2 in primary megakaryocytes by DEM treatment. DEM was added to a final concentration of 100 μM every 24 hours. CD41⁺ cells were isolated from day 1 and day 3 cultures, and nuclear extracts were prepared. Anti-Nrf2 and anti–lamin B antibodies were used. (B) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes treated with DEM. The intensities of DCFDA in the CD41⁺CD61⁺ cells are displayed in histograms. Average ROS levels obtained from the histograms of DEM-treated cells were changed to relative values against those from vehicle-treated cells. (C) Quantitative RT-PCR of platelet genes (top panel) and of cytoprotective genes (bottom panel) in DEM-treated megakaryocytes. The relative values were calculated against the values of vehicle-treated megakaryocytes. (D) Nuclear accumulation of Nrf2 in primary megakaryocytes cultured from Keap1-null fetal livers. CD41⁺ cells were isolated, and nuclear extracts were prepared. Anti-Nrf2 and anti–lamin B antibodies were used. (E) Flow cytometric analysis of intracellular ROS levels in Keap1-null megakaryocytes. The intensities of DCFDA in the CD41⁺CD61⁺ cells are displayed in histograms. Average ROS levels obtained from the histograms of Keap1-null megakaryocytes were changed to relative values of those from WT cells. Average values are shown with SD (B,E). Statistical significance of the relative ROS levels was calculated using paired t test (B,E). (F) Quantitative RT-PCR of platelet genes (top panel) and of cytoprotective genes (bottom panel) in Keap1-null megakaryocytes. The relative values were calculated against the values of WT megakaryocytes. The average values of triplicate experiments are presented, and the error bars represent SD (C,F). The Student t test was used to calculate statistical significance. *P < .002 (C,F).