M-CSF and GM-CSF Regulation of STAT5 Activation and DNA Binding in Myeloid Cell Differentiation is Disrupted in Nonobese Diabetic Mice
Figure 2
GM-CSF/M-CSF
cytokine-induced myeloid differentiation in
vitro. Bone marrow cultures
from NOD and C57BL/6 control mice were differentiated in culture using regiment
of cytokine and anticytokine blocking antibodies. Cells were treated with
either 1000?U/mL GM-CSF plus 2g/mL anti-M-CSF blocking antibodies,
with 500?U/mL M-CSF plus 2g/mL anti-GM-CSF blocking antibodies, or
with media alone for 48 hours at 3/5. Cells were stained
with anti-STAT5Ptyr-FITC (green) and anti-CD11b-PE (red) antibodies for
analysis by flow cytometry (summarized in Figure 1) and deconvolution microscopy (shown here) for the
development of cells with myeloid phenotype (CD11b+) and intracellular
expression of tyrosine phosphorylated STAT5. Background staining for
anti-STAT5Ptyr-FITC antibody was determined by parallel sample stained with a
nonspecific mouse IgG isotype control (bottom most panels). The images are composites
of 3D projection of 20 optical sections (0.2 micron each) showing both immunohistochemical
labels as well as DAPI (blue) poststaining of chromatin within the cells. Accompanying
panels show the STAT5Ptyr-FITC staining alone (green) or CD11b-PE and DAPI
staining (red/blue). Treatment regiment and the cell source strain for each
pair of cultures are listed to the left of the images. Images are representative of 5
random fields observed per sample treatment and 3 separate runs of the
experiment.
