Increased p45 and ROS levels correlate with megakaryocytic maturation. (A) Expression of p45 and Nrf2 during megakaryocytic maturation. mRNA levels (left panel) and protein levels in nuclei (right panel) are shown. CD41⁺ cells were isolated from day 1 and day 3 cultures, and total RNA and nuclear extracts were prepared. The relative values were calculated against the values of day 1 megakaryocytes (left panel). Anti-Nrf2, anti-p45, and anti–lamin B antibodies were used (right panel). (B) Flow cytometric analysis showing megakaryocytic differentiation in day 1 and day 3 cultures. Frequencies of CD41⁺CD61⁺ cells are shown in the figure (top panel). Relative frequencies of CD41⁺CD61⁺ cells were calculated against the value of day 1 culture (bottom panel). Average values are shown with SD. Statistical significance of the relative ratio of CD41⁺CD61⁺ cells was calculated using paired t test. (C) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes at day 1 and day 3. The intensities of DCFDA of CD41⁺CD61⁺ cells in panel B (gates depicted in bold rectangles) are shown in the histogram. Average ROS levels obtained from the histograms of day 3 megakaryocytes were changed to relative values against those from day 1 cells. Average values are shown with SD. Statistical significance of the relative ROS levels was calculated using paired t test. (D) Quantitative RT-PCR of platelet genes in day 1 and day 3 megakaryocytes. The relative values were calculated against the values of the day 1 sample. The average values of triplicate experiments are presented, and the error bars represent SD (A,D). The Student t test was used to calculate statistical significance. *P < .05 (A). *P < .001 (D).