Gata1 is expressed in mouse DCs. (A) Western blot of nuclear extracts of bone marrow cells grown in the presence of Flt3-L, probed with Gata1 antibody. Cells were harvested at the culture days (D) indicated. For loading control, the same blot was reprobed with Nucleophosmin (Npm1) antibody. (B) Histograms of Gata1 expression in pDCs (CD11c^medCD11b⁻B220⁺) and mDCs (CD11c⁺ CD11b⁺ B220⁻) from Flt3-L cultures, mDCs from GM-CSF cultures (CD11c^hiMHCII⁺), Eos from IL-5 cultures (CCR3⁺), and Mfs from M-CSF cultures (F4/80^hiCD11b^hi). Empty histograms show the fluorescence of the isotype control; the signals of the isotype controls depend on cell size and granularity. Filled histograms show Gata1 staining. SSC indicates side scatter. (C) Sorted DCs from Flt3-L (pDCs and mDCs) and GM-CSF (mDCs) cultures express Gata1 as shown by Western-blot analysis of nuclear extracts. Npm1 was used as loading control. Gata1 expression in DCs is lower than in bone marrow (BM) and fetal liver (FL) but clearly visible on a longer exposure (arrowheads). Sorted Eos from IL-5 cultures served as a positive control; Mfs from M-CSF cultures did not express Gata1 at detectable levels. (D) Immunofluorescence staining of cells from Flt3-L, GM-CSF, IL-5, and M-CSF cultures. The top panel shows staining with Alexa⁵⁹⁵-conjugated antirat antibody (isotype control). The bottom panel shows staining with Gata1 antibody, followed by Alexa⁵⁹⁵-conjugated antirat antibody. Nuclei are stained with DAPI (blue). Gata1 is located in the cytoplasm and nucleus of DCs and Eos. Images collected using a LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) equipped with 543-nm and 800-nm lasers. A 20×/0.5 NA was used. Pictures were taken in slides. The acquisition software used was AIM 3.2 SP2. Images were analyzed using Imaris software 4.2. (E top) Dot plots representing the sorting strategy to obtain purified CMPs from BM. (Bottom) RT-PCR analysis of Gata1 and PU.1 mRNA levels during Flt3-L–, GM-CSF–, and at the end of IL-5– and M-CSF–stimulated cultures of purified CMPs. D indicates day of culture. (F) Relative expression levels of Gata1 in vivo. Mean fluorescence intensity (MFI) of Gata1-stained samples for each cell type was divided by the MFI value of the isotype control, multiplied by 100. Eo indicates eosinophils; Lym, lymphocytes; Neu, neutrophils; imm Mo, immature monocytes; mat Mo, mature monocytes; and Mf, macrophages. Data represent average relative MFI (± SEM) calculated from 3 independent experiments. (G left) Dot plots showing the sorting strategy of pDCs and mDCs from the spleen. (Right) Sorted spleen pDCs and mDCs express Gata1 as shown by Western-blot analysis of nuclear extracts. Npm1 was used as loading control.