Nrf2 cooperates with p45 to promote megakaryocytic proliferation but does not activate platelet genes. (A) DNA content of primary megakaryocytes cultured from Keap1-null and control fetal livers (left panel). Frequencies of CD41⁺ cells containing DNA contents of 2n and between 2n and 4n are indicated. A relative number of CD41⁺ cells of each ploidy against the total CD41⁺ cells generated in the WT culture is shown in the right panel. Representative data from 2 independent experiments are shown. (B) Flow cytometric analysis of fetal liver cultures. Cells were stained with FITC-conjugated CD41 and PE-conjugated CD61. Keap1-null cells (i), Nrf2-null cells (ii), and p45-null cells (iii) were compared with control WT cells derived from their corresponding littermates. Nrf2^−/−:p45^−/− cells were compared with Nrf2-null cells (iv). Representative data are presented from more than 3 independent experiments. Frequencies of CD41⁺CD61⁺ cells are shown in the figure. Relative frequencies of CD41⁺CD61⁺ cells were calculated against the value of the WT culture (for Keap1-null, Nrf2-null, and p45-null cells) and Nrf2-null culture (for Nrf2^−/−:p45^−/− cells) (bottom panel). (C) Flow cytometric analysis showing megakaryocytic differentiation in fetal liver cultures from WT, p45-null, and p45^−/−:Keap1^−/− mice obtained from the same litter. Cells were stained with FITC-conjugated CD41 and PE-conjugated CD61. Frequencies of CD41⁺CD61⁺ cells are shown in the figure (left panel). Relative frequencies of CD41⁺CD61⁺ cells were calculated against the value of the mixture of WT and Keap1^+/− culture (right panel). Average values are shown with SD (B-C). Statistical significance of the relative ratio of CD41⁺CD61⁺ cells was calculated using paired t test (B-C). (D) Quantitative RT-PCR of platelet genes and cytoprotective genes in primary megakaryocytes cultured from Keap1^+/−, p45^−/−:Keap1^+/−, and p45^−/−:Keap1^−/− fetal livers obtained in the same litter. The relative values were calculated against the values of Keap1^+/− megakaryocytes. The average values of triplicate experiments are presented, and the error bars represent SD. The Student t test was used to calculate statistical significance. *P < .001. **P < .005.