p45 promotes ROS accumulation and increased ROS enhances platelet gene activation. (A) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes from p45-null and WT livers (top left panel), Nrf2^−/− and Nrf2^−/−:p45^−/− fetal livers (top middle panel), and Nrf2-null and WT livers (top right panel). The intensities of DCFDA in the CD41⁺CD61⁺ cells are displayed in histograms. Average ROS levels obtained from the histograms of p45-null, Nrf2^−/−:p45^−/−, and Nrf2-null cells were changed to relative values against those from the WT, Nrf2-null, and WT cells, respectively (bottom panel). Average values are shown with SD. Statistical significance of the relative ROS levels was calculated using paired t test. (B) Quantitative RT-PCR of platelet genes in primary megakaryocytes after supplementation with 10mM NAC for scavenging ROS. The relative values were calculated against the values of the vehicle-treated samples. (C) Primary megakaryocytes were fractionated into either DCFDA high or DCFDA low populations according to the fluoroprobe intensity in flow cytometry. The gates are depicted in bold rectangles. (D) Quantitative RT-PCR of platelet genes (left panel) and of p45 and Nrf2 (right panel). The CD41⁺DCFDA^high and CD41⁺DCFDA^low populations were compared. The relative values were calculated against the values of the CD41⁺DCFDA^low fraction. (E) Primary megakaryocytes cultured from p45-null and WT fetal livers were fractionated as in panel C. The intensities of DCFDA of CD41⁺ cells contained in the gate indicated with broken lines in the left panel are displayed in histograms (right panel). L and H in the right panel correspond to the gates depicted in bold rectangles in the left panel. (F) Quantitative RT-PCR of platelet genes. The CD41⁺DCFDA^high and CD41⁺DCFDA^low populations from p45-null and WT fetal livers were compared. The relative values were calculated against the values of the CD41⁺DCFDA^low fraction of WT cells. The average values of triplicate experiments are presented, and the error bars represent SD (B,D,F). The Student t test was used to calculate statistical significance. *P < .001 (B,D). **P < .05 (B,D). **P < .005 (F).