Mouse primary megakaryocytes, wild type & NF-E2p45-deficient

To identify p45 target genes, we conducted gene expression profiling with p45-null megakaryocytes cultured from E14.5 fetal liver. Many genes encoding membrane proteins and enzymes related to platelet function, including Txas, Glycoprotein 6 (Gp6) and Selectin P (Selp), were repressed in the absence of p45. Considering the similar DNA binding specificity of p45 and Nrf2 in vitro, we expected p45 to activate cytoprotective genes that are established Nrf2 targets. However, the expression of numerous detoxifying enzymes and stress-responsive genes, including NAD(P)H:quinone oxidoreductase (Nqo1), were increased in the absence of p45. To obtain wild type and p45-null fetal livers, p45+/- mice were crossed. Whole livers were recovered from mouse fetuses at E14.5 and single cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako) supplemented with 20% charcoal-stripped fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 ng/ml of recombinant human thrombopoietin. CD41+ cells were selected from a 4 day primary culture of E14.5 fetal liver cells using a MACS magnetic system (Miltenyi Biotec). The fetal liver culture with TPO described above was incubated with FITC-conjugated anti-CD41 antibody (BD Pharmingen, clone MWReg30) followed by incubation with anti-FITC microbeads. Subsequently, the microbeads were selected magnetically through MACS large cell columns (Miltenyi Biotec). Total RNA was extracted from the CD41+ cells using Isogen (Nippon Gene).