FIG. 2.

Prx2^−/− and β-thalassemic (Hbb^th3/+) mouse erythroid precursors show activation of Nrf2 and upregulation of cyoprotective genes in response to oxidative stress. (A) Nrf2 immunostaining of sorted polychromatic erythroblasts (Pop III) and orthochromatic erythroblasts (Pop IV) from bone marrow of WT Prx2^−/− and Hbb^th3/+mice. Cells were FACS sorted, cytospun onto glass slides, immunostained with anti-Nrf2 antibody, and counterstained with DAPI. Lower panel. The mean fluorescence was measured in the nucleus and cytoplasm of 30 cells using Image J software. Data are presented as means±SD (n=6); *p<0.05 compared with WT; ^{^}p<0.05 Pop III versus Pop IV. (B) Wb analysis of phospho-Nrf2 (P-Nrf2) and Nrf2 in sorted polychromatic erythroblasts (Pop III) and orthochromatic erythroblasts (Pop IV) from bone marrow of WT, Prx2^−/−, and Hbb^th3/+mice. GAPDH was used as a protein loading control. One representative gel from the other five with similar results is presented. Lower panel. Relative quantification of immunoreactivity of phospho-Nrf2 (P-Nrf2) and Nrf2 in sorted polychromatic erythroblasts (Pop III) and orthochromatic erythroblasts (Pop IV). Data are shown as means±SD (n=6). *p<0.05 compared with WT; ^{^}p<0.05 Pop III versus Pop IV. (C) RT-PCR expression of HO-1 (Hmox1) on sorted mouse polychromatic (Pop III) and orthochromatic (Pop IV) erythroblasts from bone marrow of WT, Prx2^−/−, and Hbb^3th/+mice. Experiments were performed in triplicate. Error bars represent the SDs (mean±SD); *p<0.05. The graphs were created using the Software GraphPad Prism 6. DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HO-1, heme-oxygenase-1; Nrf2, nuclear factor-erythroid 2; RT-PCR, real time polymerase chain reaction; Wb, Western blot. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars