Activation of PU.1 in human promyelocytic cells is instructive for DC fate in the absence of cytokines. Human HL-60 cells were infected with a PUER-expressing (MFG-PUERiGFP) or control retrovirus (MFG-iGFP) and individual clones were isolated (“Materials and methods”). (A) Schematic representation of used retroviral vectors. The constructs are based on an MFG vector containing an IRES-eGFP cassette. LTR indicates long terminal repeat; hER, human estrogen receptor hormone binding domain. (B) Phase contrast images of HL-60 clones expressing PUERiGFP or iGFP control retroviruses and cultured for 6 days in the absence (-βE) or presence (+βE) of 1 μM βE. (C) FACS analysis of surface antigens CD1a, CD11b, CD86, and Langerin on an HL-60 clone expressing PUERiGFP and cultured for 6 days in the absence (-βE) or presence (+βE) of 1 μM βE. (D) HL60PUER clones were induced by 1 μM βE treatment to differentiate into DCs for 48 hours then cultured in the presence or absence of 20 ng/mL LPS for an additional 48 hours under the continuous presence of βE. Expression of the activation antigens CD40 and CD83 was analyzed by FACS. The average percentage of positive cells of 6 independent clones was plotted. Error bars indicate standard error of the mean. (E) Kinetics of DC differentiation after βE treatment. PUERiGFP-expressing clones were cultured in the absence (○) or presence (▪)of1 μM βE and stained for the expression of CD1a at the indicated time points after treatment. The average percentage of CD1a-positive cells of 4 independent clones was plotted. Error bars indicate standard error of the mean. (F) FACS profiles of CD1a and GFP expression in an iGFP and PUERiGFP HL-60 clone stimulated for the indicated number of days with 1 μM βE.