PU.1 activation redirects monocytic differentiation toward DC fate. Monocyte/macrophage differentiation of HL-60 PUER clones was induced by treatment with 100 nM 1α,25-dihydroxyvitaminD3 (D3) for 20 hours. Results are representative of 2 separate experiments with 4 independent clones. (A-B) HL-60 PUER monocyte clones were either stimulated (+; ▪) or not stimulated (–; ▦) for 5 days with 1 μM βE in the continuous presence of D3. Cells were analyzed for the expression of CD11b (A) and CD1a (B) by FACS. The average for 4 independent clones is shown. Error bars indicate standard error of the mean. (C-D) Unstimulated (C) or 5-day βE-stimulated (D) HL-60 PUER monocytes maintained in the continuous presence of 100 nM D3 were analyzed for the expression of S100 protein by immunofluorescence with Alexa Red secondary antibody. Nuclei were counterstained with DAPI. (E) Primary human monocytes (Mo) were differentiated to macrophages (MΦ) or DCs with M-CSF or GM-CSF and IL-4, respectively. Western blot analysis for PU.1 expression was performed on total cell extracts from 2 different donors after 7 days of culture. (F) Monocytes were subjected to DC differentiation conditions as in panel E, collected at different time points of culture, and analyzed for expression of PU.1 by Western blot. Tubulin expression was used as internal control for protein concentration of Western extracts in panels E and F. (G) Monocytes in panel F were analyzed for expression of the DC marker CD1a and the monocyte/macrophage marker CD14 antigens by FACS staining. (H) Kinetics of CD1a, CD14, and PU.1 levels during monocyte-to-DC differentiation. CD1a (▴) and CD14 (▪) are represented as percent positive cells and PU.1 levels (○) as percent of maximal expression (16 hours) normalized to tubulin.