PU.1 expression is tightly regulated during FL erythropoiesis. (A) Erythroid precursors are GFP-positive (GFP⁺). The 13.5-dpc Lin^– GFP⁺ and Lin^– GFP^– cells were purified and cultured in erythroid medium. Starting from 104 cells, the number of cells in each culture was quantified daily. Lin^–: B220^–Gr1^–TER119^–-CD3^–F4/80^–CD16/32^–NK1.1^–. (B) GFP expression is lost in culture. The 13.5-dpc PU.1^+/G and PU.1G/G FL cells were cultured. GFP expression in the CD71^loTER119^– and CD71^+/hiTER119^– populations was measured after indicated times of culture. (C) PU.1 is expressed in “GFP-negative” erythroid progenitors and down-regulated in differentiated erythrocytes. Immature CD71⁺TER119^– and mature TER119⁺ cells were purified after 7 days of culture (these cells are GFP-negative by flow cytometry) and analyzed for PU.1 and GFP expression by RT-PCR. The residual PU.1 and GFP mRNAs detected in TER119⁺ cells are probably due to contaminating TER119^– cells (about 5%). (D) Erythroid progenitors synthesize PU.1. Western blot of nuclear extracts from FL cells cultured for 7 days, and depleted of TER119⁺ cells, total BM, and MEL cells using antibodies for PU.1 and β-actin. All experiments were performed in erythroid medium and are representative of more than 3 experiments.